Multi-target RNA inhibitor

Abstract

A multi-target RNA inhibitor, relating to the field of nucleic acid drugs, and having specific structures as shown in the following general formulae Ia and Ib. X1 and X2 are orthogonal reactive groups, and the orthogonal reactive groups comprise, but are not limited to: one or more of amino groups, amido groups, carboxylic acid groups, azides, alkynes, propargyl groups, dibenzocyclooctyne (DBCO), phosphoesters, maleimide groups, aminooxy groups, N-hydroxysuccinimide (NHS), PNP, TFP, PFP, bromo groups, aldehydes, carbonate groups, tosyl groups, tetrazines, trans-cyclooctene (TCO), hydrazides, hydroxyl groups, disulfides and o-pyridyl disulfide groups, bicyclo[6.1.0]nonyne (BCN), and free or protected thiol groups. Za, Zb, Zc and Zd are delivery ligands or H, k is an integer from 0 to 4; and L1, L2, L3 and L4 are connecting chains or bonds. The present invention can achieve the goal of delivering a multi-target siRNA drug or a plurality of same-target siRNA drugs to the same cells.

Description

A multi-target RNA inhibitor TECHNICAL FIELD

[0001] The present application relates to the field of nucleic acid therapy, in particular to a multi-target RNA inhibitor. BACKGROUND

[0002] RNAi

[0003] RNAi (RNA interference) was discovered in 1998 by Andrew Z. Fire et al. when they performed antisense RNA inhibition experiments in Caenorhabditis elegans and named the process RNA interference. This discovery was ranked as one of the top ten scientific advances of 2001 by the journal Science and was ranked as the top scientific advance of 2002. Since then, siRNA as a potential gene therapy drug has received widespread attention due to its mechanism of action. In 2006, Andrew Z. Fire and Craig C. Mello won the Nobel Prize in Physiology or Medicine for their contributions to the study of the mechanism of RNA interference. RNAi is triggered by double-stranded RNA (dsRNA) in many organisms, including animals, plants and fungi. In the process of RNA inhibition, a nucleic acid endonuclease called "Dicer" cuts or "dices" long dsRNA into small fragments of 21-25 nucleotides long. These small fragments, called small interfering RNA (siRNA), have an antisense strand (Guide strand) loaded onto an Argonaute protein (AGO2). AGO2 loading occurs in the RISC-loading complex, which is a ternary complex composed of Argonaute protein, Dicer and dsRNA binding protein (referred to as TRBP). During the loading process, the passenger strand is cleaved by AGO2 and is discharged. Then, AGO2 binds the antisense strand to mRNA containing a completely complementary sequence, and then catalyzes the cleavage of these mRNAs, causing the mRNA to split and lose the function of a translation template, thereby preventing the synthesis of related proteins. After cleavage, the cleaved mRNA is released, and the RISC-loading complex loaded with the antisense strand is recycled for another round of cleavage.

[0004] According to statistics, among the disease-related proteins in the human body, more than 80% of the proteins cannot be targeted by the current conventional small molecule drugs and biological macromolecular preparations, which belong to the undruggable proteins. The gene therapy aiming to treat diseases through gene expression, silencing and other functions is considered by the industry as the third generation of therapeutic drugs after chemical small molecule drugs and biological macromolecular drugs. This therapy achieves treatment of diseases at the gene level and is not restricted by undruggable proteins. As the most mainstream type of RNAi technology in gene therapy, RNAi technology treats diseases at the mRNA level, which has higher efficiency than the treatment of chemical small molecule drugs and biological macromolecular drugs at the protein level. Using RNAi technology, specific siRNA sense and antisense chain sequences with high specificity and good inhibition effect can be designed according to specific gene sequences, and these single-chain sequences are synthesized by solid phase, and then the sense and antisense chains are paired into siRNA in a specific annealing buffer according to the base pairing principle, and finally delivered to the corresponding target in the body through the carrier system, degrading the target mRNA and destroying the function of the target mRNA as a translation template, thereby preventing the synthesis of related proteins.

[0005] Delivery system of siRNA

[0006] siRNA is unstable in blood and tissues and is easily degraded by nucleases. In order to improve the stability of siRNA, the sense and / or antisense chain of siRNA can be modified, but these chemical modifications only provide limited protection against nuclease degradation and can ultimately affect the activity of siRNA. Therefore, a corresponding delivery system is also needed to ensure that siRNA safely and efficiently crosses the cell membrane. Since the siRNA molecule has a large molecular weight and carries a large number of negative charges, and has high water solubility, it cannot successfully cross the cell membrane to reach the inside of the cell.

[0007] Liposome basic structure is composed of hydrophilic core and phospholipid bilayer, with similar biological membrane phospholipid bilayer, has very high biocompatibility, so the liposome once become the most popular, the most widely used siRNA carrier. Liposome-mediated siRNA delivery mainly wraps siRNA into liposome, protects siRNA from nuclease degradation, improves the efficiency of siRNA through the cell membrane barrier, thereby promoting the absorption of cells. For example, anionic liposome, pH-sensitive liposome, immunoliposome, membrane fusion liposome (fusogenic liposome) and cationic lipids, etc., although some progress has been made, but the liposome itself can easily trigger an inflammatory response, before administration must use a variety of antihistamines and hormones such as cyproheptadine and dexamethasone and other drugs to reduce the possible acute inflammatory response, so in the actual clinical application is not suitable for all treatment areas, especially some chronic disease treatment areas, long-term use of potential accumulation of toxicity is a potential safety hazard, so we need a safer and more effective carrier system to deliver siRNA.

[0008] Asialoglycoprotein receptor (ASGPR) in the liver, is a receptor specifically expressed in hepatocytes, is a highly efficient endocytic receptor. Because of the physiological conditions in vivo, various glycoproteins are exposed to the penultimate galactose residues after enzymatic or acid hydrolysis of sialic acid, so the ASGPR specifically binds to galactosyl sugar, so it is also called galactose-specific receptor. Galactose, galactosamine, N-acetylgalactosamine and other monosaccharide and polysaccharide molecules have high affinity for ASGPR. The main physiological function of ASGPR is to mediate the clearance of asialoglycoprotein, lipoprotein and other substances in the blood, and is closely related to the occurrence and development of viral hepatitis, cirrhosis, liver cancer and other liver diseases. The discovery of the characteristics of ASGPR plays an important role in the diagnosis and treatment of liver diseases (Ashwell G, Harford J, Carbohydrate specific Receptors of the Liver, Ann Rev Biochem 1982 51:531-554). Liver disease treatment drugs containing galactose or galactosamine and their derivatives in the structure can specifically bind to ASGPR, thereby having active liver targeting, without the need for other carrier systems for delivery.

[0009] Delivery modes include ligand compounds that specifically bind to other receptors, such as: alpha v beta 6 receptor, auxiliary membrane permeable molecules that help oligonucleotide internalization into cells of neuronal origin, polypeptide molecules, such as: RGD polypeptides that specifically bind to integrin family alpha v beta 3, cell-penetrating peptides, etc., antibody molecules; these ligands or auxiliary membrane permeable molecules are coupled to oligonucleotides through orthogonal reactive groups and linking chains. Representative examples of orthogonal reactive groups include, but are not limited to: amino, amide, carboxylic acid, azide, alkyne, propargyl, BCN (bicyclo[6.1.0]nonyne), DBCO (dibenzocyclooctyne), thiol, maleimide, aminooxy, N-hydroxysuccinimide (NHS) or other activated esters (e.g. PNP, TFP, PFP), bromo, aldehyde, carbonate, tosylate, tetrazine, trans-cyclooctene (TCO), hydrazide, hydroxyl, disulfide and orthopyridyl disulfide groups, free or protected thiol, etc. DBCO series products include DBCO-acid, DBCO-amine, DBCO-PEG-COOH, DBCO-PEG-amine, and DBCO-labeled Biotin, fluorescent dyes and biochemical small molecules, etc. that can be used for further connection.

[0010] There is a market need for a delivery mode that can simultaneously deliver multi-target oligonucleotides, and the present application solves such a problem. SUMMARY

[0011] Provided herein is an RNA inhibitor that couples multiple oligonucleotides to ligands through orthogonal reactive groups, linking chains, which can achieve the delivery of multi-target oligonucleotides.

[0012] An RNA inhibitor that contains double-stranded small interfering nucleic acids for one or more targets in its chemical structure, and the specific structure is as follows: general formula Ia, Ib:

[0013] X1and X2are orthogonal reactive groups, including but not limited to one or more of the following: amino, amido, carboxylic acid, azide, alkyne, propargyl, DBCO dibenzocyclooctyne, phosphine ester, maleimide, aminooxy, N-hydroxysuccinimidyl NHS, PNP, TFP, PFP, bromo, aldehyde, carbonate, tosylate, tetrazine, trans-cyclooctene TCO, hydrazide, hydroxyl, disulfide and orthopyridyl disulfide groups, BCN bicyclo[6.1.0]nonyne, free or protected thiol; it is noted that this is not an exhaustive list, and the present application is intended to protect the use of linker groups to string together multiple siRNA targeting sites, and to increase delivery via delivery ligands, and any approach that uses the inventive concept of multiple targeting siRNA delivery is within the scope of the present application.

[0014] Z a , Z b , Z c , Z d is a delivery ligand or H;

[0015] k is an integer from 0 to 4;

[0016] L1, L2, L3, L4 are a linking chain or bond, the linking chain having the structure of the general formula: -A-(B-A)n-:

[0017] each B is independently a substituted or unsubstituted C1-10alkylene, C2-10alkenylene, C2-10alkynylene, C3-10cycloalkylene, C6-14arylene, heteroarylene, or heterocycloalkylene;

[0018] each A is an independent bond, -C(O)-, -C(O)O-, -C(O)NR 1b -, -NR 1b C(O)-, -(OCH2CH2) n -, -(CH2CH2O) n -, -(CH2OCH2) n -, -C(O)S-, -C(NR 1a )NR 1b -, -C(S)-, -C(S)O-, -C(S)NR 1b -, -C(R 1a )=NO-, -O-, -OC(O)O-, -OC(O)NR 1b -, -OC(O)S-, -OC(NR 1a )NR 1b -, -OC(S)O-, -OC(S)NR 1b -, -OS(O)-, -OS(O)2-, -OS(O)NR 1b-,-OS(O)2NR 1b -,-NR 1b -,-NR 1a C(O)NR 1b -,-NR 1a C(O)S-,-NR 1a C(NR 1d )NR 1b -,-NR 1a C(S)NR 1b -,-NR 1a S(O)NR 1b -,-NR 1a S(O)2NR 1b -,-P(O2)O-,-P(O)(S)O-,-S-,-SS-,-S(O)-,-S(O)2-,-S(O)NR 1b -, or -S(O)2NR 1b -;

[0019] M can be selected from: substituted or unsubstituted alkyl or heteroalkyl groups, specifically from -C(O)-, -C(O)O-, -C(O)NR1b-, -NR1bC(O)-, -(CH2)nC(O)NH(CH2)mNH C(O)(CH2)n-, -(OCH2CH2)n-, -(CH2CH2O)n-, -(CH2OCH2)n-, -(CH2)n C(O)NH(CH2CH2O)m(CH2)nNH C(O)(CH2)n-, -(CH2)nC(O)N(CH2)mCHC(O)NH(CH2)mNH C(O)CH(CH2)mN C(O)(CH2)n-,-C(O)S-,-C(NR1a)NR1b-,-C(S)-,-C(S)O-,-C(S)NR1b-,-C(R1a)=NO-,-O-,-OC(O)O-,-OC (O)NR1b-,-OC(O)S-,-OC(NR1a)NR1b-,-OC(S)O-,-OC(S)NR1b-,-OS(O)-,-OS(O)2-,-OS(O)NR1b-,-OS(O )2NR1b-,-NR1b-,-NR1aC(O)NR1b-,-NR1aC(O)S-,-NR1aC(NR1d)NR1b-,-NR1aC(S)NR1b-,-NR1aS(O)NR1b -,-NR1aS(O)2NR1b-,-P(O2)O-,-P(O)(S)O-,-S-,-SS-,-S(O)-,-S(O)2-,-S(O)NR1b-, or -S(O)2NR1b-;-;

[0020] Each R1a ,R 1b ,R 1c , and R 1d Independently: (i) hydrogen or deuterium; or substituted or unsubstituted (ii) C1-6 alkyl, C1-6 heteroalkyl, C2-6 alkenyl, C2-6 ynyl, C3-10 cycloalkyl, C6-14 aryl, C7-15 aryl, heteroaryl or heterocyclic.

[0021] n is an integer from 0 to 10; m is an integer from 0 to 12;

[0022] Delivery ligands include one or more of the following: ligand compounds targeting the ASGPR receptor, ligand compounds targeting αvβ6 integrin, membrane-penetrating molecule, peptide molecule, or antibody molecule.

[0023] The delivery ligand is conjugated to the terminal nucleotide of the siRNA, the internal nucleotide of the siRNA, or the linker strand.

[0024] In some embodiments, the aforementioned RNA inhibitor I) uses general formula (II) to tandemly connect the 5' ends of the sense strand of a double-stranded small interfering nucleic acid, wherein a delivery ligand is conjugated to the 3' end or internally of the sense strand, or a delivery ligand is conjugated to the 3' end or internally of the antisense strand paired with the sense strand; schematic diagrams of general formulas (IIa, IIb) are shown below:

[0025] The 3' ends of the sense strands of double-stranded small interfering nucleic acids are tandemly linked using general formulas (Ⅲa, Ⅲb), wherein a delivery ligand is conjugated to the 5' end or internally of the sense strand, or a delivery ligand is conjugated to the 5' end or internally of the antisense strand paired with the sense strand; a schematic diagram of general formulas (Ⅲa, Ⅲb) is shown below:

[0026] The 3' ends of the antisense strands of double-stranded small interfering nucleic acids are tandemly linked using general formulas (Ⅳa,Ⅳb), and a delivery ligand is conjugated to the 5' end or internally of the sense strand that pairs with the antisense strand; a schematic diagram of general formulas (Ⅳa,Ⅳb) is shown below:

[0027] Wherein, X1 and X2 are orthogonal reactive groups, which include: amino, amide, carboxylic acid, azide, alkyne, propargyl, DBCO dibenzocyclooctylene, maleimide, aminooxy, N-hydroxysuccinimide NHS, PNP, TFP, PFP, bromine, aldehyde, carbonate, toluenesulfonate, tetrazine, transcyclooctene TCO, hydrazide, hydroxyl, disulfide and o-pyridyl disulfide, BCN bicyclo[6.1.0]nonyne, free or protected thiols, one or more of these groups;

[0028] M is:

[0029] ss is the sense strand and as is the antisense strand;

[0030] Z a , Z b , Z1, Z2, Z3, Z4, Z5, Z6 are delivery ligands or H;

[0031] L1, L2, L3, L4 are linkers or bonds, said linkers having the structure of the general formula: -A-(B-A)n-:

[0032] each B is independently a substituted or unsubstituted C1-10alkylene, C2-10alkenylene, C2-10alkynylene, C3-10cycloalkylene, C6-14arylene, heteroarylene, or heterocycloalkylene;

[0033] each A is an independent bond, -C(O)-, -C(O)O-, -C(O)NR 1b -, -NR 1b C(O)-, -(OCH2CH2) n -, -(CH2CH2O) n -, -(CH2OCH2) n -, -C(O)S-, -C(NR 1a )NR 1b -, -C(S)-, -C(S)O-, -C(S)NR 1b -, -C(R 1a ) = NO-, -O-, -OC(O)O-, -OC(O)NR 1b -, -OC(O)S-, -OC(NR 1a )NR 1b -, -OC(S)O-, -OC(S)NR 1b -, -OS(O)-, -OS(O)2-, -OS(O)NR 1b -, -OS(O)2NR 1b -, -NR 1b -, -NR 1a C(O)NR 1b -, -NR 1a C(O)S-, -NR 1a C(NR 1d )NR 1b -, -NR 1a C(S)NR 1b -, -NR 1a S(O)NR 1b -, -NR 1a S(O)2NR 1b-,-P(O2)O-,-P(O)(S)O-,-S-,-SS-,-S(O)-,-S(O)2-,-S(O)NR 1b -, or -S(O)2NR 1b -;

[0034] M can be selected from: substituted or unsubstituted alkyl groups, heteroalkyl groups, and can be selected from –C(O)–, –C(O)O–, –C(O)NR. 1b –,–NR 1b C(O)–,–(CH2)nC(O)NH(CH2)mNH C(O)(CH2)n–,–(OCH2CH2)n–,–(CH2CH2O)n–,–(CH2OCH2)n–,–(CH2)n C(O)NH(CH2CH2O)m(CH2)nNH C(O)(CH2)n–,–(CH2)nC(O)N(CH2)mCHC(O)NH(CH2)mNH C(O)CH(CH2)mN C(O)(CH2)n–,–C(O)S–,–C(NR 1a )NR 1b –,–C(S)–,–C(S)O–,–C(S)NR 1b –,–C(R 1a )=NO–,–O–,–OC(O)O–,–OC(O)NR 1b –,–OC(O)S–,–OC(NR 1a )NR 1b –,–OC(S)O–,–OC(S)NR 1b –,–OS(O)–,–OS(O)2–,–OS(O)NR 1b –,–OS(O)2NR 1b –,–NR 1b –,–NR1aC(O)NR 1b –,–NR 1a C(O)S–,–NR 1a C(NR 1d )NR 1b –,–NR 1a C(S)NR 1b –,–NR 1a S(O)NR 1b –,–NR 1a S(O)2NR 1b –,–P(O2)O–,–P(O)(S)O–,–S–,–S–S–,–S(O)–,–S(O)2–,–S(O)NR 1b –, or –S(O)2NR 1b –;

[0035] each R 1a , 1b , 1c , and R 1d , independently, is: (i) hydrogen or deuterium; or (ii) substituted or unsubstituted C1-6alkyl, C1-6heteroalkyl, C2-6alkenyl, C2-6alkynyl, C3-10cycloalkyl, C6-14aryl, C7-15aralkyl, heteroaryl, or heterocyclyl;

[0036] n is an integer from 0 to 10; m is an integer from 1 to 12;

[0037] the delivery ligand comprises one or more of: a ligand compound targeting the ASGPR receptor, a ligand compound targeting the ανβ6integrin, a helper transmembrane molecule, a polypeptide molecule, or an antibody molecule;

[0038] the delivery ligand is conjugated to an end nucleotide of the siRNA, to an internal nucleotide of the siRNA, or to a linking chain.

[0039] In some embodiments, the aforementioned one RNA inhibitor, X1and X2are azide and DBCO, respectively, and the compound has the following general formula, General Formula Va, Vb, Via, VIb, VIc, VI d:

[0040] wherein, R1-R 10 independently hydrogen, halogen, hydroxyl, or alkoxy, wherein the alkyl portion of the alkoxy is C1, C2, C3, C4, C5, C7alkyl;

[0041] Za, Zb, Zc, Zd are delivery ligands or H;

[0042] k is an integer from 0 to 4;

[0043] L1, L2, L3, L4 are linking chains or bonds, the linking chain having the following general structure: -A-(B-A)n-:

[0044] each B is independently substituted or unsubstituted C1-10alkylene, C2-10alkenylene, C2-10alkynylene, C3-10cycloalkylene, C6-14arylene, heteroarylene, or heterocyclylene;

[0045] each A is independently a bond, -C(O)-, -C(O)O-, -C(O)NR 1b -, 1b C(O)-, - (OCH2CH2) n -, n - (CH2CH2O) -, - (CH2OCH2)n -C(O)S-, -C(NR 1a )NR 1b -C(S)-, -C(S)O-, -C(S)NR 1b -C(R 1a )=NO-, -O-, -OC(O)O-, -OC(O)NR 1b -OC(O)S-, -OC(NR 1a )NR 1b -OC(S)O-, -OC(S)NR 1b -OS(O)-, -OS(O)2-, -OS(O)NR 1b -OS(O)2NR 1b -NR 1b -NR 1a C(O)NR 1b -NR 1a C(O)S-, -NR 1a C(NR 1d )NR 1b -NR 1a C(S)NR 1b -NR 1a S(O)NR 1b -NR 1a S(O)2NR 1b -P(O2)O-, -P(O)(S)O-, -S-, -S-S-, -S(O)-, -S(O)2-, -S(O)NR 1b -, or -S(O)2NR 1b -;

[0046] M can be selected from substituted or unsubstituted alkyl, heteroalkyl, can be selected from -C(O)-, -C(O)O-, -C(O)NR 1b -, -NR 1b C(O)-, -(CH2)nC(O)NH(CH2)mNH C(O)(CH2)n-, -(OCH2CH2)n-, -(CH2CH2O)n-, -(CH2OCH2)n-, -(CH2)n C(O)NH(CH2CH2O)m(CH2)nNH C(O)(CH2)n-, -(CH2)nC(O)N(CH2)mCHC(O)NH(CH2)mNH C(O)CH(CH2)mN C(O)(CH2)n-, -C(O)S-, -C(NR 1a )NR 1b -, -C(S)-, -C(S)O-, -C(S)NR 1b -, -C(R1a ) = NO-, -O-, -OC(O)O-, -OC(O)NR 1b -, -OC(O)S-, -OC(NR 1a )NR 1b -, -OC(S)O-, -OC(S)NR 1b -, -OS(O)-, -OS(O)2-, -OS(O)NR 1b -, -OS(O)2NR 1b -, -NR 1b -, -NR1aC(O)NR 1b -, -NR 1a C(O)S-, -NR 1a C(NR 1d )NR 1b -, -NR 1a C(S)NR 1b -, -NR 1a S(O)NR 1b -, -NR 1a S(O)2NR 1b -, -P(O2)O-, -P(O)(S)O-, -S-, -S-S-, -S(O)-, -S(O)2-, -S(O)NR 1b -, or -S(O)2NR 1b -;

[0047] wherein M is preferably selected from:

[0048] each R 1a , R 1b , R 1c , and R 1d is independently: (i) hydrogen or deuterium; or (ii) substituted or unsubstituted C1-6alkyl, C1-6heteroalkyl, C2-6alkenyl, C2-6alkynyl, C3-10cycloalkyl, C6-14aryl, C7-15arylalkyl, heteroaryl, or heterocyclyl;

[0049] n is an integer from 1 to 10; m is an integer from 1 to 12;

[0050] The delivery ligand is not limited, as one example, the delivery ligand includes, but is not limited to, one or more of a ligand compound targeting ASGPR receptor, a ligand compound targeting ανβ6integrin, a facilitator transmembrane molecule, a polypeptide molecule, or an antibody molecule;

[0051] The conjugation position of the delivery ligand is not limited, as an example, the delivery ligand is conjugated on the end nucleotide of the siRNA, on the internal nucleotide of the siRNA or on the connecting chain.

[0052] In some embodiments, the aforementioned one RNA inhibitor, X1, X2 are respectively: thiol and olefin, the specific general formula is as follows: the compound of general formula VII, VIII:

[0053] Wherein, R1-R5 are independently hydrogen, halogen, hydroxyl or alkoxy, wherein the alkyl part of the alkoxy is C1, C2, C3, C4, C5, C7 alkyl;

[0054] Za, Zb, Zc, Zd are delivery ligands or H;

[0055] K is an integer from 0 to 4;

[0056] L1, L2, L3, L4 are connecting chains or bonds, the connecting chain has the structure of the following general formula: -A-(B-A)n-:

[0057] Each B is independently substituted or unsubstituted C1-10alkylene, C2-10alkenylene, C2-10alkynylene, C3-10cycloalkylene, C6-14arylene, heteroarylene, or heterocycloalkylene;

[0058] Each A is an independent bond, -C(O)-, -C(O)O-, -C(O)NR 1b -,-NR 1b C(O)-, -(OCH2CH2) n -,-(CH2CH2O) n -,-(CH2OCH2) n -,-C(O)S-,-C(NR 1a )NR 1b -,-C(S)-,-C(S)O-,-C(S)NR 1b -,-C(R 1a )=NO-,-O-,-OC(O)O-,-OC(O)NR 1b -,-OC(O)S-,-OC(NR 1a )NR 1b -,-OC(S)O-,-OC(S)NR 1b -,-OS(O)-,-OS(O)2-,-OS(O)NR 1b -,-OS(O)2NR 1b -,-NR 1b -,-NR 1a C(O)NR 1b -,-NR1a C(O)S-,-NR 1a C(NR 1d )NR 1b -,-NR 1a C(S)NR 1b -,-NR 1a S(O)NR 1b -,-NR 1a S(O)2NR 1b -,-P(O2)O-,-P(O)(S)O-,-S-,-SS-,-S(O)-,-S(O)2-,-S(O)NR 1b -, or -S(O)2NR 1b -;

[0059] Each R 1a ,R 1b ,R 1c , and R 1d Independently: (i) hydrogen or deuterium; or substituted or unsubstituted (ii) C1-6 alkyl, C1-6 heteroalkyl, C2-6 alkenyl, C2-6 ynyl, C3-10 cycloalkyl, C6-14 aryl, C7-15 aryl, heteroaryl or heterocyclic.

[0060] n is an integer from 1 to 10;

[0061] The delivery ligand is not limited, but as an example, the delivery ligand includes, but is not limited to, one or more of the following: ligand compounds targeting the ASGPR receptor, ligand compounds targeting αvβ6 integrin, membrane-penetrating molecule, peptide molecule or antibody molecule.

[0062] The location of the delivery ligand conjugate is not limited; as an example, the delivery ligand conjugate is attached to the terminal nucleotide of the siRNA, the internal nucleotide of the siRNA, or the linker strand.

[0063] The aforementioned RNA inhibitor, X1 and X2, are BCN and an azide compound, respectively, with the following general formula: General Formula IX:

[0064] in,

[0065] Za, Zb, Zc, and Zd are delivery ligands or H;

[0066] k is an integer between 0 and 4;

[0067] L1, L2, L3, and L4 are connecting chains or keys, and the connecting chains have the following general formula: -A-(BA)n-:

[0068] each B is independently substituted or unsubstituted C1-10alkylene, C2-10 alkenylene, C2-10 alkynylene, C3-10 cycloalkylene, C6-14 arylene, heteroarylene, or heterocycloalkylene;

[0069] each A is an independent bond, -C(O)-, -C(O)O-, -C(O)NR 1b -,-NR 1b C(O)-, -(OCH2CH2) n -,-(CH2CH2O) n -,-(CH2OCH2) n -,-C(O)S-, -C(NR 1a )NR 1b -,-C(S)-, -C(S)O-, -C(S)NR 1b -,-C(R 1a ) = NO-, -O-, -OC(O)O-, -OC(O)NR 1b -,-OC(O)S-, -OC(NR 1a )NR 1b -,-OC(S)O-, -OC(S)NR 1b -,-OS(O)-, -OS(O)2-, -OS(O)NR 1b -,-OS(O)2NR 1b -,-NR 1b -,-NR 1a C(O)NR 1b -,-NR 1a C(O)S-, -NR 1a C(NR 1d )NR 1b -,-NR 1a C(S)NR 1b -,-NR 1a S(O)NR 1b -,-NR 1a S(O)2NR 1b -,-P(O2)O-, -P(O)(S)O-, -S-, -S-S-, -S(O)-, -S(O)2-, -S(O)NR 1b -, or -S(O)2NR 1b -;

[0070] each R 1a ,R 1b ,R 1c , and R 1dindependently: (i) hydrogen or deuterium; or (ii) substituted or unsubstituted C1-6alkyl, C1-6heteroalkyl, C2-6alkenyl, C2-6alkynyl, C3-10cycloalkyl, C6-14aryl, C7-15aralkyl, heteroaryl, or heterocyclyl;

[0071] n is an integer from 1 to 10;

[0072] The delivery ligand includes one or more of: a ligand compound targeting the ASGPR receptor, a ligand compound targeting the ανβ6integrin, a facilitator transmembrane molecule, a polypeptide molecule, or an antibody molecule;

[0073] The delivery ligand is conjugated to an end nucleotide of the siRNA, to an internal nucleotide of the siRNA, or to a linking chain.

[0074] One of the aforementioned RNA inhibitors, X1, X2are: azide and phosphine ester, the specific general formula is as follows: the compound of general formula X:

[0075] wherein R1-R3are independently hydrogen, halogen, hydroxyl, or alkoxy, wherein the alkyl portion of the alkoxy is C1, C2, C3, C4, C5, C7alkyl;

[0076] Za, Zb, Zc, Zdare delivery ligands or H;

[0077] k is an integer from 0 to 4;

[0078] L1, L2, L3, L4are linking chains or bonds, the linking chain has the structure of the following general formula: -A-(B-A)n-:

[0079] Each B is independently substituted or unsubstituted C1-10alkylene, C2-10alkenylene, C2-10alkynylene, C3-10cycloalkylene, C6-14arylene, heteroarylene, or heterocyclylene;

[0080] Each A is independently a bond, -C(O)-, -C(O)O-, -C(O)NR 1b -,-NR 1b C(O)-,-(OCH2CH2) n -,-(CH2CH2O) n -,-(CH2OCH2) n -,-C(O)S-,-C(NR 1a )NR 1b -,-C(S)-,-C(S)O-,-C(S)NR 1b -,-C(R 1a )=NO-,-O-,-OC(O)O-,-OC(O)NR 1b-OC(O)S-, -OC(NR 1a )NR 1b -OC(S)O-, -OC(S)NR 1b -OS(O)-, -OS(O)2-, -OS(O)NR 1b -OS(O)2NR 1b -NR 1b -NR 1a C(O)NR 1b -NR 1a C(O)S-, -NR 1a C(NR 1d )NR 1b -NR 1a C(S)NR 1b -NR 1a S(O)NR 1b -NR 1a S(O)2NR 1b -P(O2)O-, -P(O)(S)O-, -S-, -S-S-, -S(O)-, -S(O)2-, -S(O)NR 1b -, or -S(O)2NR 1b -;

[0081] each R 1a , R 1b , R 1c , and R 1d is independently: (i) hydrogen or deuterium; or (ii) substituted or unsubstituted C1-6alkyl, C1-6heteroalkyl, C2-6alkenyl, C2-6alkynyl, C3-10cycloalkyl, C6-14aryl, C7-15aralkyl, heteroaryl, or heterocyclyl;

[0082] n is an integer from 1 to 10;

[0083] The delivery ligand includes one or more of a ligand compound targeting the ASGPR receptor, a ligand compound targeting the ανβ6integrin, a facilitator transmembrane molecule, a polypeptide molecule, or an antibody molecule;

[0084] The delivery ligand is conjugated to an end nucleotide of the siRNA, to an internal nucleotide of the siRNA, or to a linking chain.

[0085] One of the aforementioned RNA inhibitors, X1and X2are each: TCO and tetrazine, specifically the following compound of Formula XI:

[0086] wherein,

[0087] Za, Zb, Zc, Zdare delivery ligands or H;

[0088] k is an integer from 0 to 4;

[0089] L1, L2, L3, L4 are linking chains or bonds, said linking chains having the structure of the general formula: -A-(B-A)n-:

[0090] each B is independently a substituted or unsubstituted C1-10alkylene, C2-10 alkenylene, C2-10alkynylene, C3-10 cycloalkylene, C6-14 arylene, heteroarylene, or heterocycloalkylene;

[0091] each A is an independent bond, -C(O)-, -C(O)O-, -C(O)NR 1b -,-NR 1b C(O)-, -(OCH2CH2) n -,-(CH2CH2O) n -,-(CH2OCH2) n -,-C(O)S-, -C(NR 1a )NR 1b -,-C(S)-, -C(S)O-, -C(S)NR 1b -,-C(R 1a ) = NO-, -O-, -OC(O)O-, -OC(O)NR 1b -,-OC(O)S-, -OC(NR 1a )NR 1b -,-OC(S)O-, -OC(S)NR 1b -,-OS(O)-, -OS(O)2-, -OS(O)NR 1b -,-OS(O)2NR 1b -,-NR 1b -,-NR 1a C(O)NR 1b -,-NR 1a C(O)S-, -NR 1a C(NR 1d )NR 1b -,-NR 1a C(S)NR 1b -,-NR 1a S(O)NR 1b -,-NR 1a S(O)2NR 1b -,-P(O2)O-, -P(O)(S)O-, -S-, -S-S-, -S(O)-, -S(O)2-, -S(O)NR 1b -, or -S(O)2NR 1b -;

[0092] each R 1a each R 1b each R 1c each R 1d independently: (i) hydrogen or deuterium; or (ii) substituted or unsubstituted C1-6alkyl, C1-6heteroalkyl, C2-6alkenyl, C2-6alkynyl, C3-10cycloalkyl, C6-14aryl, C7-15arylalkyl, heteroaryl, or heterocyclyl;

[0093] n is an integer from 1 to 10;

[0094] The delivery ligand includes one or more of: a ligand compound targeting ASGPR receptor, a ligand compound targeting ανβ6 integrin, a helper transmembrane molecule, a polypeptide molecule, or an antibody molecule;

[0095] The delivery ligand is conjugated to an end nucleotide of the siRNA, an internal nucleotide of the siRNA, or a linking chain.

[0096] The foregoing RNA inhibitor, each L1, L2, L3, L4independently is: wherein p is an integer from 0 to 200, and m is an integer from 0 to 12.

[0097] The foregoing RNA inhibitor, the ligand compound targeting ASGPR receptor has the structure of Z R -L5-O-P(O)(O)-O-.

[0098] The foregoing RNA inhibitor, Z R has the structure as shown below:

[0099] The foregoing RNA inhibitor, L5is a linking chain, and has the structure of -A-(B-A)n-:

[0100] each B independently is substituted or unsubstituted C1-10alkylene, C2-10alkenylene, C2-10alkynylene, C3-10cycloalkylene, C6-14arylene, heteroarylene, or heterocyclylene;

[0101] each A is independently a bond, -C(O)-, -C(O)O-, -C(O)NRib-, -NRibC(O)-, -(OCH2CH2)n-, -(CH2CH2O)n-, -(CH2OCH2)n-, -C(O)S-, -C(NRla)NRib-, -C(S)-, -C(S)O-, -C(S)NRib-, -C(Rla)=NO-, -O-, -OC(O)O-, -OC(O)NRib-, -OC(O)S-, -OC(NRla)NRib-, -OC(S)O-, -OC(S)NRib-, -OS(O)-, -OS(O)2-, -OS(O)NRib-, -OS(O)2NRib-, -NRib-, -NRlaC(O)NRib-, -NRlaC(O)S-, -NRlaC(NRld)NRib-, -NRlaC(S)NRib-, -NRlaS(O)NRib-, -NRlaS(O)2NRib-, -P(O2)O-, -P(O)(S)O-, -S-, -S-S-, -S(O)-, -S(O)2-, -S(O)NRib-, or -S(O)2NRib-;

[0102] each Rla, Rlb, Rlc, and Rldis independently: (i) hydrogen or deuterium; or (ii) substituted or unsubstituted C1-6alkyl, C1-6heteroalkyl, C2-6alkenyl, C2-6alkynyl, C3-10cycloalkyl, C6-14aryl, C7-15aralkyl, heteroaryl, or heterocyclyl;

[0103] n is an integer from 1 to 10.

[0104] In some embodiments, the foregoing RNA inhibitor, each L5is independently: wherein p is an integer from 0 to 200, and m is an integer from 0 to 12. The above examples are not exhaustive, and the structure of linker is not limited as long as it is a structure that can be achieved by synthetic chemistry.

[0105] As an example, the foregoing RNA inhibitor, the structure of the ligand compound targeting ASGPR receptor is:

[0106] The foregoing RNA inhibitor, the structure of the auxiliary membrane-penetrating molecule is:

[0107] wherein x is an integer from 1 to 10;

[0108] each G is substituted or unsubstituted: C2-30alkyl, C2-30alkene, C2-30heteroalkyl, C2-30alkenyl, C2-30alkynyl, C2-30cycloalkyl, C2-30aryl, C2-30aryl, C2-30heteroaryl, or C2-30heterocyclyl;

[0109] I is C or N;

[0110] each L 4a、 L 4b having the structure: -A-(B-A)n-:

[0111] each B is independently a bond, substituted or unsubstituted C1-10alkylene, C2-10alkenylene, C2-10alkynylene, C3-10cycloalkylene, C6-14arylene, heteroarylene, or heterocyclylene;

[0112] each A is independently a bond, -C(O)-, -C(O)O-, -C(O)NR1b-, -NR1bC(O)-, -(OCH2CH2)n-, -(CH2CH2O)n-, -(CH2OCH2)n-, -C(O)S-, -C(NR1a)NR1b-, -C(S)-, -C(S)O-, -C(S)NR1b-, -C(R1a)=NO-, -O-, -OC(O)O-, -OC(O)NR1b-, -OC(O)S-, -OC(NR1a)NR1b-, -OC(S)O-, -OC(S)NR1b-, -OS(O)-, -OS(O)2-, -OS(O)NR1b-, -OS(O)2NR1b-, -NR1b-, -NR1aC(O)NR1b-, -NR1aC(O)S-, -NR1aC(NR1d)NR1b-, -NR1aC(S)NR1b-, -NR1aS(O)NR1b-, -NR1aS(O)2NR1b-, -P(O2)O-, -P(O)(S)O-, -S-, -S-S-, -S(O)-, -S(O)2-, -S(O)NR1b-, or -S(O)2NR1b-;

[0113] each R1a, R1b, R1c, and R1d is independently: (i) hydrogen or deuterium; or (ii) substituted or unsubstituted C1-6alkyl, C1-6heteroalkyl, C2-6alkenyl, C2-6alkynyl, C3-10cycloalkyl, C6-14aryl, C7-15aryl, heteroaryl, or heterocyclyl;

[0114] n is an integer from 1 to 10. Without limitation, the structure of the linker is not limited to those described herein, but rather any structure that can be synthesized by synthetic chemistry can be used in the present application.

[0115] The aforementioned RNA inhibitor, the G structure includes: The structure of the lipid molecule is not limited, as long as the molecule can help the membrane to be suitable for the present application.

[0116] The aforementioned RNA inhibitor, characterized in that,

[0117] As some examples, the aforementioned RNA inhibitor, an oligonucleotide, targets the following genes: C3, C5, CFB, SAA, TTR, RSV, PDGFβ gene, Erb-B gene, Src gene, CRK gene, APP, STAT3, survivin gene, MAPT, PCSK9, ANGPTL3, APOC3, LPA, AGT, ALDH2, PNPLA3, KHK, HBV, LDHA, factor VII, Eg5, TPX2, apoB, MY. C, HPV, MKK4, CLAUDINE-1, SNCA, ACVRIC (ALK-7), Her2 / Neu gene, SID-1, MIG-12, MASP2, INFRSF12B, AT3, repressor factor VII, PTEN, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1 / 2 gene, PCNA (p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, Cyclin Genes including D, VEGF, EGFR, Cyclin A, cyclin E, WNT-1, β-catenin, c-MET, PKC, NFKB, Klf-1, bcl11a, serpinal, tmprss6, TTR, INHBE, CYP2A6, TM6SF2, Aha, IKK-b, CCR5, HAO1, SORT1, XBP1, topoisomerase I, topoisomerase IIα, p73, p21 (WAF1 / CIP1), p27 (KIP1), PPM1D, RAS, caveolin I, MIB I, MTAI, M68, tumor suppressor genes, and p53 tumor suppressor gene. The above examples are not exhaustive; the targeted mRNA targets are not limited. Attached Figure Description

[0118] Figure 1. Inhibitory effect of DDP0404-DS004 on PCSK9 protein expression in cynomolgus monkey serum.

[0119] Figure 2. Inhibitory effect of DDP0404-DS004 on APOC3 protein expression in cynomolgus monkey serum.

[0120] Figure 3. Inhibitory effect of DDP0404-DS004 on LDL-C levels in cynomolgus monkey serum.

[0121] Figure 4. Inhibitory effect of DDP0404-DS004 on serum TG levels in cynomolgus monkeys.

[0122] Figure 5. Inhibitory effect of DDP0404-DS00501 on PCSK9 protein expression in cynomolgus monkey serum.

[0123] Figure 6D DDP0404-DS00501 inhibitory effect on Lp a protein expression level in serum of cynomolgus monkey

[0124] Figure 7 DDP0404-DS00501 inhibitory effect on TG level in serum of cynomolgus monkey

[0125] Figure 8 DDP0404-DS006 inhibitory effect on PCSK9 protein expression level in serum of cynomolgus monkey

[0126] Figure 9 DDP0404-DS006 inhibitory effect on ANGPTL3 protein expression level in serum of cynomolgus monkey

[0127] Figure 10 DDP0404-DS006 inhibitory effect on LDL-C level in serum of cynomolgus monkey

[0128] Figure 11 DDP0404-DS006 inhibitory effect on TC level in serum of cynomolgus monkey

[0129] DETAILED DESCRIPTION

[0130] The present application will be readily understood by the following detailed description in conjunction with the accompanying drawings, and the foregoing information. Other advantages of the application will be realized and appreciated by the skilled person once aware of the application as described herein.

[0131] TERMS

[0132] In the present application, the term "angiopoietin-like protein 3" can be used interchangeably with the term "complement system", examples of complement system mRNA sequences are readily available using publicly available databases, for example, GenBank, UniProt, OMIM and the Macaca Genome Project website.

[0133] The term "CFB" refers to complement factor B, mRNA sequences of which can be found in, for example, GenBank NM_001710.5; 001710; cynomolgus monkey NC_041757.1, mouse NM_001142706 and NM_008198; rat NM_212466.3.

[0134] The term "C3" refers to complement C3, mRNA sequences of which can be found in, for example, GenBank NM_000064.4; cynomolgus monkey NC_000019.10; mouse NM_009778; rat NM_016994.

[0135] The term "C5" refers to complement C5, mRNA sequences of which can be found in, for example, GenBank NM_001735.3; mouse NM_013485; rat NM_057146.

[0136] The term "Lp a", "LPA", "lipoprotein(a)" or "apolipoprotein(a)" is the name of the gene encoding apolipoprotein(a) (apo(a)), the human LPA mRNA sequence can be found, for example, in GenBank NM_005577.4.

[0137] The term "PCSK9" refers to proprotein convertase subtilisin / kexin type 9 (PCSK9),

[0138] The term "APOC3 (apolipoprotein C-III)" is the gene encoding apolipoprotein C3, examples of APOC3 mRNA sequences are readily available using, for example, the publicly available databases of GenBank, UniProt, and OMIM. The term "APOC3" includes human APOC3, cynomolgus APOC3, rhesus APOC3, mouse APOC3, rat (Rattus norvegicus) APOC3, rabbit (Oryctolagus cuniculus) APOC3, and the like; as long as the organism has an APOC3 gene, it is within the scope of the present disclosure. The mRNA sequence of human APOC3 can be found, for example, in GenBank NM_000040.3. Cynomolgus APOC3, the amino acid and its complete coding sequence can be found, for example, in GenBank Accession No. GI:544489959 (XM_05579730.1); rhesus APOC3, the amino acid and its complete coding sequence can be found, for example, in GenBank Accession No. GI:297269260 (XM_001090312.2); mouse APOC3, the amino acid and its complete coding sequence can be found, for example, in GenBank Accession No. GI:577019555 (NM_023114.4); rat (Rattus norvegicus) APOC3, the amino acid and its complete coding sequence can be found, for example, in GenBank Accession No. GI:402534545 (NM_012501.2); and rabbit (Oryctolagus cuniculus) APOC3, GenBank Accession No. GI:655601498 (XM_002708371.2).

[0139] The term "TAU" refers to microtubule-associated protein tau (MAPT), the mRNA sequence of which can be found, for example, in siRNA targeting the human TAU (MAPT) gene (human: NCBI refseq ID GenBank NM_NM_016841.5):

[0140] The term "APP" refers to amyloid precursor protein (APP), the mRNA sequence of which can be found, for example, in siRNA targeting the human APP gene (human: NCBI refseq ID GenBank NM_201414.3)

[0141] Indicators of "inhibition of expression of CFB" include: inhibition of mRNA levels of the CFB gene, inhibition of protein levels of the CFB gene, and any level of inhibition, and can also be: CH50 activity as a measure of total hemolytic complement, AH50, which measures hemolytic activity of the alternative complement pathway, and / or lactate dehydrogenase (LDH) levels as a measure of intravascular hemolysis, and / or hemoglobin levels; and can also measure levels of C3, C9, C5, C5a, C5b, and soluble C5b-9 complex to evaluate CFB expression levels.

[0142] Indicators of "inhibition of expression of C3" include: C3 assays, total complement assays, immune complex assays, C3 plasma concentration assays, C3 fixed antibody method tests; C3 assays can be inhibition of mRNA levels of the C3 gene, or inhibition of protein levels of the C3 gene, and any level of inhibition; C3 assays can be performed with other complement components to assess the function of the entire complement system. This typically includes measuring C4 concentration as well as CH50 and other indicators; immune complex assays: immune complexes are structures composed of antibodies and antigens, which form in certain autoimmune diseases. Serum immune complex assays can be used to detect these immune complexes and assess whether activation of the immune system is occurring, leading to a decrease in C3; C3 plasma concentration assays: assess the function of the complement system by measuring the concentration of C3 in the plasma. Normally, the concentration of C3 should be within the normal range; C3 fixed antibody method: this method is used to measure the ability of C3 to bind to specific antigens to assess the function of the complement system. It can also be measuring indicators related to C3 gene levels such as: CH50 activity as a measure of total hemolytic complement, AH50, which measures hemolytic activity of the alternative complement pathway, and / or lactate dehydrogenase (LDH) levels as a measure of intravascular hemolysis, and / or hemoglobin levels. C3 expression can also be evaluated by measuring levels of CFB, C9, C5, C5a, C5b, and soluble C5b-9 complex.

[0143] Indicators of "inhibition of expression of C5" include: inhibition of mRNA levels of the C5 gene, inhibition of protein levels of the C5 gene, and any level of inhibition, and can also be: CH50 activity as a measure of total hemolytic complement, AH50, which measures hemolytic activity of the alternative complement pathway, and / or lactate dehydrogenase (LDH) levels as a measure of intravascular hemolysis, and / or hemoglobin levels; and can also measure levels of C3, C9, C5, C5a, C5b, and soluble C5b-9 complex to evaluate CFB expression levels.

[0144] The term "APP" refers to the amyloid precursor protein (APP) gene, which encodes an integral membrane protein expressed in neurons and glia. Amyloid precursor protein-APP is an integral membrane protein expressed in a variety of tissues and concentrated at synapses of neurons. APP is believed to be closely associated with Alzheimer's disease. APP can be cleaved by alpha-, beta-, and gamma- proteases; wherein sequential action of beta- and gamma- proteases can cause APP to be cleaved to produce Aβ. Aβ can cause the formation of senile plaques in the brain and apoptosis of neural cells, Aβ(1-42) (also known as Aβ42) and Aβ(1-40) (also known as Aβ40) are commonly found as the major proteins in the beta amyloid plaque deposits that have long been described as being associated with the development and progression of Alzheimer's disease (AD) in affected individuals.

[0145] The term "microtubule-associated protein Tau (MAPT)" refers to the microtubule-associated protein tau (MAPT) gene, which encodes the microtubule-associated protein Tau, is a member of the microtubule-associated gene family, located in chromosomal region 17q21.31 (base pairs 45,894,382 to 46,028,334 on chromosome 17). The MAPT gene consists of 16 exons. Alternative mRNA splicing produces six MAPT isoforms, totaling 352-441 amino acids. In three of the six MAPT isoforms, the microtubule-binding domain of MAPT contains three repeat segments, while the corresponding domain in the other three MAPT isoforms contains four repeat segments. The microtubular system is a component of the cytoskeleton of nerve cells and can be involved in a variety of cellular functions. Microtubules are composed of tubulin and microtubule-associated proteins, with Tau protein being the most abundant microtubule-associated protein. The cellular function of Tau protein in the normal brain is to bind to tubulin and promote its polymerization to form microtubules; to bind to the formed microtubules to maintain their stability by reducing the dissociation of tubulin molecules, and to induce bundling of microtubules. Tau protein is a phospho-protein, with 2-3 phosphate groups per Tau protein molecule in the normal mature brain. In the brains of patients with Alzheimer's disease (senile dementia), Tau protein is abnormally hyperphosphorylated, with 5-9 phosphate groups per Tau protein molecule, and loses its normal biological function. Enzyme-linked immunosorbent assay (ELISA) can be used to determine Tau protein in the plasma and cerebrospinal fluid (CSF) of AD patients. Studies have shown that the level of Tau protein in the CSF of AD patients is significantly higher than that in the normal and non-neurological disease patient groups of the same age. The use of increased Tau protein levels in the CSF to diagnose AD has a sensitivity of 82% and a specificity of 70%. If the increase in Tau protein levels in the CSF and the decrease in β-AP42 levels are measured simultaneously, the specificity of AD diagnosis can reach 70-90%.

[0146] In the present application, "target gene" refers to an RNA gene to be silenced, which can be mRNA or tRNA or viral RNA. In some embodiments, the "target gene" targets the following genes: C3, C5, CFB, SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene, APP, STAT3, Survivin gene, TAU, PCSK9, ANGPTL3, APOC3, LPA, AGT, ALDH2, PNPLA3, KHK, HBV, LDHA, Factor VII, Eg5, TPX2, apoB, MYC, HPV, MKK4, CLAUDINE-1, SNCA, ACVRIC (ALK-7), Her2 / Neu gene, SID-1, MIG-12, MASP2, INFRSF12B, AT3, Inhibitor Factor VII, PTEN, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1 / 2 gene, PCNA (p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, Cyclin D gene, VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene, PKC gene, NFKB gene, Klf-1, bcl11a, serpinal, tmprss6, TTR, INHBE, CYP2A6, TM6SF2, Aha, IKK-b, CCR5, HAO1, SORT1 gene, XBP1 gene, Topoisomerase I gene, Topoisomerase II alpha gene, p73 gene, p21 (WAF1 / CIP1) gene, p27 (KIP1) gene, PPM1D gene, RAS gene, caveolin I gene, MIB I gene, MTAI gene, M68 gene, tumor suppressor gene, p53 tumor suppressor gene; this is not an exhaustive list, and the selection of target genes is not limited, and can be applied to the present application.

[0147] In the present application, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of a complement system gene, including mRNA that is a product of RNA processing of a primary transcription product. In some embodiments, the target portion of the sequence will be at least long enough to serve as a substrate for RNA inhibitor-directed degradation at or near the portion of the nucleotide sequence of an mRNA molecule formed during transcription of a complement system gene. The length of the "target sequence" is typically about 15-30 nucleotides.

[0148] In the present application, the term "nucleic acid drug" refers to a class of drugs that includes oligonucleotides, ribozymes, plasmids, immunostimulatory nucleic acids, antagomirs, antimir, microRNA mimics, supermirs, U1 adaptors, and aptamers. These nucleic acids exert their effects through a variety of mechanisms. Oligonucleotide drugs, in general, reduce the expression level of a specific protein through the process of RNA interference (RNAi).

[0149] In the present application, the term "oligonucleotide" refers to a short chain of nucleic acids composed of deoxyribonucleotides or ribonucleotides. Commonly used oligonucleotides include antisense oligonucleotides (ASO), siRNA (small interfering RNA), microRNA, and nucleic acid aptamers.

[0150] In the present application, the term "RNA inhibitor" refers to an agent comprising RNA as defined by the term herein, and which mediates the targeted cleavage of an RNA transcript through the RNA-induced silencing complex (RISC) pathway. Sequence-specific degradation of mRNA is directed via a process known as RNA interference, which modulates (e.g., inhibits) the expression of a complement system gene in a cell (e.g., a cell in a subject, such as a mammalian subject).

[0151] In some embodiments, the RNA inhibitor can be a single-stranded siRNA (ssRNA inhibitor) introduced into a cell or organism to inhibit a target mRNA (i.e., a complement system gene). The single-stranded RNA inhibitor binds to the endonuclease Argonaute 2 within RISC, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15 to 30 nucleotides in length and are chemically modified.

[0152] In some embodiments, the "RNA inhibitor" used in the present application is a double-stranded RNA, and is referred to in the present application as a "double-stranded RNA inhibitor," a "double-stranded RNA (dsRNA, DS) molecule," a "dsRNA agent," or a "dsRNA." The term "dsRNA" refers to a complex of ribonucleic acid molecules having a duplex structure comprising two antiparallel and substantially complementary nucleic acid strands, referred to as having "sense" and "antisense" orientation with respect to a target mRNA. In some embodiments of the present application, double-stranded RNA (dsRNA) triggers the degradation of a target mRNA through a post-transcriptional gene silencing mechanism referred to in the present application as RNA interference or RNAi.

[0153] The duplex structure can be of any length that triggers specific degradation of the complement system mRNA by the RISC pathway, and can range in length from about 15 to 36 base pairs, e.g., about 15-30 base pairs, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 36 base pairs in length. In certain embodiments, the RNA inhibitor of the application is a 15-30 nucleotide dsRNA that interacts with the target sequence to direct cleavage of the complement system mRNA.

[0154] Generally, the sense and antisense strands of a dsRNA molecule are predominantly ribonucleotides, but can also include one or more non-ribonucleotides, e.g., deoxyribonucleotides or modified nucleotides, as described in detail herein. In addition, the RNA inhibitors contemplated by the present disclosure can include ribonucleotides having chemical modifications, and can have modified nucleotides at various regions. The term "modified nucleotide" as used herein means a nucleotide independently having a modified sugar moiety, a modified internucleotide linkage, or a modified nucleobase, or any combination thereof. Thus, the term "modified nucleotide" encompasses substitution, addition, or removal of, e.g., a functional group or atom, to the internucleotide linkage, sugar moiety, or nucleobase. Modifications suitable for use in the RNA inhibitors of the present application include all types of modifications disclosed herein or known in the art.

[0155] In the present disclosure, the term "nucleotide sequence" generally refers to a string or sequence of nucleotides, whether modified or unmodified, described by a string of letters using standard nucleotide nomenclature and the symbol table for modified nucleotides described herein. The nucleotide sequences described herein are polymers made up of linked monomers via phosphodiester bonds (or related structural variants or synthetic analogs thereof), including naturally occurring nucleotide polymers, but it is understood that the scope of the term also includes various analogs, including but not limited to: peptide nucleic acids (PNAs), phosphoramidates, phosphorothioates, methylphosphonates, and 2'-O-methyl ribonucleic acids, etc. Generally, there are about 15-30 nucleotides, but the term can also refer to molecules of any length.

[0156] In some embodiments, the nucleotide sequence comprises one or more unmodified ribonucleosides (RNA) and / or unmodified deoxyribonucleosides (DNA) and / or one or more modified nucleotides. The term "modified nucleotide sequence" generally means a string or sequence of nucleotides comprising at least one modification and / or at least one modified internucleotide linkage.

[0157] In the present application, the term "modified nucleotide" generally means a nucleotide comprising at least one chemical modification compared to a naturally occurring RNA or DNA nucleotide. For example, a 2' deoxythymidine nucleotide, a 2' O-methyl modified nucleotide, a 2' fluoro modified nucleotide, a 2' deoxy modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2' amino modified nucleotide, a 2' O long chain alkyl modified nucleotide (e.g., hexadecyl), a morpholino nucleotide, a phosphoramidate nucleotide, a non-natural nucleobase nucleotide, a 5' thiophosphoester nucleotide, and a nucleotide linked to a cholesterol derivative or a dodecanoic acid didecanamide group.

[0158] A modified nucleotide comprises a modified sugar group and / or a modified nucleobase.

[0159] In the present application, the term "nucleobase" or "base" generally means a heterocyclic pyrimidine or purine compound that is a component of all nucleic acids and includes adenine, guanine, cytosine, thymine, and uracil. A nucleotide can include a modified nucleotide or nucleotide mimic, an abasic (Abasic) or surrogate moiety. The term "unmodified nucleobase" or "naturally occurring nucleobase" generally means a naturally occurring heterocyclic nucleobase of RNA or DNA: the purine bases adenine and guanine; and the pyrimidine bases thymine, cytosine, and uracil. A "modified nucleobase" generally means any nucleobase that is not a naturally occurring nucleobase.

[0160] In the present application, the term "sugar group" generally means a naturally occurring sugar group of a nucleotide or a modified sugar group. The term "naturally occurring sugar group" generally means a furanoribose group as found in naturally occurring RNA or a deoxyfuranoribose group as found in naturally occurring DNA. A "modified sugar group" means a substituted sugar group or sugar surrogate, for example, a fluoro or methoxy substitution at the 2' position of the sugar group.

[0161] In the present application, the term "internucleotide linkage" generally means a covalent linkage between adjacent nucleotides in a nucleotide sequence. A "naturally occurring internucleotide linkage" means a 3' to 5' phosphodiester linkage. A "modified internucleotide linkage" means any internucleotide linkage other than a naturally occurring internucleotide linkage.

[0162] In the present application, the term "antisense strand" (AS) generally refers to a strand of an RNA inhibitor (e.g., a dsRNA) that includes a region of substantial complementarity to a target sequence. As used in the present application, the term "region of complementarity" generally refers to a region on an antisense strand that is substantially complementary to a sequence defined in the present application (e.g., a target sequence).

[0163] In the present application, the term "sense strand" (S) generally refers to the strand of an RNA inhibitor (e.g., dsRNA) that includes a region that is substantially complementary to a region of the "antisense strand" (AS). The "sense" strand is sometimes referred to as the "sense" strand, the "passenger" strand, or the "anti-guide" strand. With the sequence of the sense strand, the antisense strand targets the desired mRNA, while the sense strand can target a different target or be degraded. Thus, if the antisense strand is incorporated into RISC, the correct target is targeted. Incorporation of the sense strand can lead to off-target effects. These off-target effects can be limited by using modifications on the sense strand or using a 5' end cap.

[0164] The loading of the siRNA duplex into the ago protein must first be initiated by the recognition of the 5' end of the antisense strand, which is a prerequisite for the placement of the rest of the duplex into the AGO protein's nucleic acid binding channel. The MID domain of the AGO protein recognizes the nucleotide at the 5' end, upon recognition, the siRNA duplex is loaded into the AGO protein's nucleic acid binding channel to form pre-RISC. After the loading of the siRNA duplex into the nucleic acid binding channel, the pre-RISC expels the sense strand (passenger strand) and the remaining antisense strand (guide strand) forms the RISC.

[0165] In the present application, the term "complementary" refers to the ability of two nucleotide sequences to hybridize under certain conditions, form base pair hydrogen bonds, and form a duplex or double helix structure. As the antisense strand of an RNA inhibitor hybridizes to the sense strand of an RNA inhibitor or the mRNA of the complement system to form Watson-Crick base pairs or non-Watson-Crick base pairs, and includes natural or modified nucleotides or nucleotide mimics. "Complementary" does not necessarily have nucleobase complementarity on every nucleoside. Instead, some mismatches can be tolerated.

[0166] In the present application, the term "mismatch", when the complementary region is not completely complementary to the target sequence, the mismatch can be in the core region or the terminal region. Generally, the most tolerated mismatches are in the terminal region, for example, within 5, 4, 3, or 2 nucleotides at the 5' end and / or 3' end, and no more than 3 mismatches.

[0167] As shown in the results of Gu S, Jin L, Zhang F, Huang Y, Grimm D, Rossi JJ, Kay MA. Thermodynamic stability of small hairpin RNAs highly influences the loading process of different mammalian Argonautes. Proc Natl Acad Sci USA 2011, 108: 9208-9213, any factor that influences the thermodynamic stability of the duplex, such as mismatches and non-Watson-Crick base pairs, favors the ejection of the sense strand by pre-RISC to form RISC.

[0168] In the present application, the term "pharmaceutically acceptable" generally refers to one or more nontoxic substances with essentially no effective biological activity at the concentrations at which they are administered. Exemplary materials suitable for such use are well known to those skilled in the art. Such formulations will typically contain salts, excipients, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. Such pharmaceutically acceptable formulations will also typically include compatible solid or liquid fillers, diluents, or encapsulating material which are suitable for administration to humans. When used in medicine, salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts can conveniently be used to prepare pharmaceutically acceptable salts. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, boric, formic, malonic, succinic, and the like. Pharmaceutically acceptable salts can also be prepared as alkali metal or alkaline earth salts, such as sodium, potassium or calcium salts.

[0169] The term "pharmaceutically acceptable excipient" refers to a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, diluent, solvent, or encapsulation material. In one embodiment, each component is "pharmaceutically acceptable" in that it is compatible for use with other ingredients of a drug formulation, and suitable for use in contact with the tissue or organ of a subject (e.g., a human) without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications commensurate with a reasonable benefit / risk ratio. See Remington: The Science and Practice of Pharmacy, 23rdEd.; Adejare Ed. Academic Press, 2020; Handbook of Pharmaceutical Excipients 9thEd.; Sheski et al. Eds.; Pharmaceutical Press, 2020; Handbook of Pharmaceutical Additives, 3rdEd.; Ash and Ash Eds. Synapse Information Resources, 2007; Pharmaceutical Preformulation and Formulation, 1stEd.; Gibson Ed. CRC Press, 2015.

[0170] In the present application, the term "ligand" generally refers to any compound or molecule that is capable of covalently or otherwise chemically binding to a biologically active substance, such as a dsRNA. In some embodiments, a ligand is capable of directly or indirectly interacting with another compound, e.g., a receptor, which can be present on the surface of a cell, or alternatively can be an intracellular and / or intercellular receptor, the interaction of the ligand with the receptor can result in a biochemical reaction, or can simply be a physical interaction or binding.

[0171] In the present application, the term "delivery ligand" can be a targeting ligand that targets a specific receptor, can be a "membrane-facilitating molecule" that is non-targeting but facilitates entry of the inhibitor into the contents of the cell membrane, or can be a "lipid nanoparticle" that encapsulates the nucleic acid drug and enables membrane penetration.

[0172] In the present application, the term "targeting ligand" alters the distribution, targeting, or stability of the RNA inhibitor by introducing a delivery ligand in the vector that targets a tissue receptor. For example, a targeting ligand provides enhanced affinity for a selected target (e.g., a molecule, a cell or cell type, a compartment (e.g., a cellular or organ compartment, a body tissue, an organ, or a region)) compared to the species in the absence of the ligand. This includes, without limitation: an antibody, an antigen, folate, a receptor ligand, a carbohydrate, an aptamer, an integrin receptor ligand, a chemokine receptor ligand, transferrin, biotin, a serotonin receptor ligand, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands. Among these, the carbohydrate includes, but is not limited to, D-galactose, N-acetyl-D-galactosamine (GalNAc), D-mannose, lactose, N-acetyl-glucosamine, fucose, glycosylated polyamino acids, lectins, or analogs of these compounds.

[0173] In the present application, the term "lipid nanoparticle" or "LNP" generally refers to a vesicle comprising a lipid layer that encapsulates a pharmacologically active molecule, such as a dsRNA. LNPs are described, for example, in Chinese Patent No. CN103189057B, the entire contents of which are incorporated herein by reference.

[0174] In the present application, the term "assisting membrane permeation molecule" refers to a molecule that assists the nucleic acid drug to enter a cell, usually a lipid-soluble molecule, including but not limited to: saturated or unsaturated alkane chain, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-0(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl, palmitic acid, myristic acid, 03-(oleoyl)etolic acid, 03-(oleoyl)cholenic acid, ibuprofen, naproxen, dimethoxybenzophenone or phenoxazine, amphiphilic peptides, cell penetration peptides, endosomolytic / fusogenic peptides, alkylating agents, phosphates, amines, sulfhydryls, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport / absorption facilitators (e.g. naproxen, aspirin, vitamin E), synthetic ribozymes (e.g. imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, AP, hormones and hormone receptors, lectins, carbohydrates, multivalent carbohydrates, vitamins (e.g. vitamin A, vitamin E, vitamin K, vitamin B). The chain length of the saturated or unsaturated alkane chain can be 4-30 carbons, preferably 6-22 carbons. The alkane can be a straight chain alkyl or alkenyl group. The alkane can also contain functional groups such as hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azido and alkyne groups.

[0175] The delivery ligand can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low density lipoprotein (LDL), or globulin); a carbohydrate (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine, or hyaluronic acid); or a lipid. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid.

[0176] In the present application, the term "click reaction" is introduced in Gramlich et al., Angew. Chem. Int. Ed., 2008, 47, 8350-8358; Amblard et al., Chem. Rev., 2009, 109, 4207-4220, which provide an overview of "click" chemistry and oligonucleotide synthesis. U.S. Patent 6,737,236 discloses a cycloaddition reaction for conjugation of biomolecules. For example, the 1,3-dipolar cycloaddition conjugation between alkyne and azide (later described as click chemistry, Sharpless et al. Angew. Chem. Int. Ed. 40:2004 (2001)) produces a substituted triazine as a moiety of the conjugation product.

[0177] Bioorthogonal chemical reactions are chemical reactions that can occur in biological systems without interfering with natural biochemical processes. For more information, please refer to the article published by Professor Fan Xinyuan and his team in Chemical Journal in 2021. Representative examples of orthogonal reactive groups include but are not limited to: amino, amide, carboxylic acid, azide, alkyne, propargyl, BCN (bicyclo[6.1.0]nonyne), DBCO (dibenzocyclooctyne), thiol, maleimide, aminooxy, N-hydroxysuccinimide (NHS) or other activated esters (such as PNP, TFP, PFP), bromo, aldehyde, carbonate, tosylate, tetrazine, trans-cyclooctene (TCO), hydrazide, hydroxyl, disulfide and ortho-pyridyl disulfide groups, free or protected thiols, etc. DBCO products include DBCO-acid, DBCO-amine, DBCO-PEG-COOH, DBCO-PEG-amine, and DBCO-labeled Biotin, fluorescent dyes, and biochemical small molecules, which can be used for further connection.

[0178] Biocompatibility of orthogonal reactive groups:

[0179] DBCO (dibenzocyclooctyne) is biodegradable and is used in the synthesis of conjugates (ADCs) as a degradable ADC linker. This degradable property allows DBCO to break down under certain conditions, releasing the target drug or biomolecule. BCN (boron carbon nitride) is biodegradable, and BCN has excellent biocompatibility, mechanical stability, and biodegradability, which makes it have potential application value in the field of biomedicine. Tetrazine is a biodegradable compound. This property makes tetrazine have important applications in biomedical research, especially in the synthesis of antibody conjugate drugs (ADCs). As a degradable ADC linker, tetrazine can break under certain conditions, releasing drug molecules, achieving targeted therapy, and reducing damage to normal cells. In addition, the tetrazine group in tetrazine has high reactivity and can undergo bioorthogonal reactions with trans-cyclooctene (TCO), such as inverse electron demand Diels-Alder reaction (iEDDA). This reaction is particularly useful in bioorthogonal chemistry because it can be performed without interfering with other functional groups in the biological system, allowing for precise labeling and tracking of biomolecules. Tetrazine also shows potential in inhibiting the growth, metastasis, and recurrence of immune rejection tumors in anti-tumor efficacy evaluation.

[0180] The term "alkyl" refers to linear or branched saturated monovalent hydrocarbon radicals, wherein the alkyl is optionally substituted with one or more substituents Q described herein. For example, C1-6alkyl refers to a linear saturated monovalent hydrocarbon radical of 1 to 6 carbon atoms or a branched saturated monovalent hydrocarbon radical of 3 to 6 carbon atoms. In certain embodiments, the alkyl is a linear saturated monovalent hydrocarbon radical of 1 to 20 (C1-20), 1-15 (C1-15), 1-10 (C1-10), or 1-6 (C1-6) carbon atoms, or a branched saturated monovalent hydrocarbon radical of 3 to 20 (C3-20), 3-15 (C3-15), 3-10 (C3-10), or 3-6 (C3-6) carbon atoms. As used herein, linear C1-6and branched C3-6alkyl groups are also referred to as "lower alkyl." Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl (including all isomeric forms such as, for example, n-propyl and isopropyl), butyl (including all isomeric forms such as, for example, n-butyl, isobutyl, sec-butyl, and t-butyl), pentyl (including all isomeric forms such as, for example, n-pentyl, isopentyl, sec-pentyl, neopentyl, and t-pentyl), and hexyl (including all isomeric forms such as, for example, n-hexyl, isohexyl, and sec-hexyl).

[0181] The terms "alkylene" and "alkanediyl" are used interchangeably herein to refer to a linear or branched saturated divalent hydrocarbon radical, wherein the alkanediyl is optionally substituted with one or more substituents Q described herein. For example, C1-6alkanediyl refers to a linear saturated divalent hydrocarbon radical of 1 to 6 carbon atoms or a branched saturated divalent hydrocarbon radical of 3 to 6 carbon atoms. In certain embodiments, the alkanediyl is a linear saturated divalent hydrocarbon radical having 1 to 30 (C1-30), 1 to 20 (C1-20), 1 to 15 (C1-15), 1 to 10 (C1-10), or 1 to 6 (C1-6) carbon atoms, or a branched saturated divalent hydrocarbon radical having 3 to 30 (C3-30), 3 to 20 (C3-20), 3 to 15 (C3-15), 3 to 10 (C3-10), or 3 to 6 (C3-6) carbon atoms. Examples of the alkanediyl group include, but are not limited to: methanediol, ethanediyl (including all isomeric forms, e.g., ethane-1,1-diyl ethane-1,2-diyl), propanediol (including all isomeric forms, e.g., propane-1,1-diyl, propane-1,2-diyl, and propane-1,3-diyl), butanediol (including all isomeric forms, e.g., butane-1,1-diyl, butane-1,2-diyl, butane-1,3-diyl, and butane-1,4-diyl), pentanediol (including all isomeric forms, e.g., pentane-1,1-diyl, pentane-1,2-diyl, pentane-1,3-diyl, pentane-1,5-diyl), and hexanediol (including all isomeric forms, e.g., hexane-1,1-diyl, hexane-1,2-diyl, hexane-1,3-diyl, and hexane-1,6-diyl). Examples of substituted alkanediyl groups include, but are not limited to: -C(O)CH2-, -C(O)(CH2)2-, -C(O)(CH2)3-, -C(O)(CH2)4-, -C(O)(CH2)5-, -C(O)(CH2)6-, -C(O)(CH2)7-, -C(O)(CH2)8-, -C(O)(CH2)9-, -C(O)(CH2)10-, -C(O)CH2C(O)-, -C(O)(CH2)2C(O)-, -C(O)(CH2)3C(O)-, -C(O)(CH2)4C(O)-, or -C(O)(CH2)5C(O)-.

[0182] The term "heteroalkyl" refers to a linear or branched saturated monovalent hydrocarbon radical comprising one or more heteroatoms in its backbone, each heteroatom being independently selected from O, S, and N. Heteroalkyl is optionally substituted with one or more substituents Q described herein. For example, Ci-6heteroalkyl refers to a linear saturated monovalent hydrocarbon radical of 1 to 6 carbon atoms or a branched saturated monovalent hydrocarbon radical of 3 to 6 carbon atoms. In certain embodiments, heteroalkyl is a linear saturated monovalent hydrocarbon radical of 1 to 20 (Ci-20), 1 to 15 (Ci-15), 1 to 10 (Ci-10), or 1 to 6 (Ci-6) carbon atoms or a branched saturated monovalent hydrocarbon radical of 3 to 20 (C3-20), 3 to 15 (C3-15), 3 to 10 (C3-10), or 3 to 6 (C3-6) carbon atoms. Examples of heteroalkyl include, but are not limited to, -OCH3, -OCH2CH3, -CH2OCH3, -NHCH3, -ONHCH3, -NHOCH3, -SCH3, -CH2NHCH2CH3, and -NHCH2CH2CH3. Examples of substituted heteroalkyl include, but are not limited to, -CH2NHC(O)CH3and -NHC(O)CH2CH3.

[0183] The terms "heteroalkylene" and "heteroalkanediyl" are used interchangeably herein to refer to a linear or branched saturated divalent hydrocarbon radical containing one or more heteroatoms in its backbone, each heteroatom being independently selected from O, S, and N. For example, C1-6heteroalkyl refers to a linear saturated divalent hydrocarbon radical of 1 to 6 carbon atoms or a branched saturated divalent hydrocarbon radical of 3 to 6 carbon atoms. In certain embodiments, the heteroalkyl radical is a linear saturated divalent hydrocarbon radical of 1 to 20 (C1-20), 1 to 15 (C1-15), 1 to 10 (C1-10), or 1 to 6 (C1-6) carbon atoms, or a branched saturated divalent hydrocarbon radical of 3 to 20 (C3-20), 3 to 15 (C3-15), 3 to 10 (C3-10), or 3 to 6 (C3-6) carbon atoms. Examples of heteroalkyl radicals include, but are not limited to, -CH2O-, -(CH2)2O-, -(CH2)3O-, -(CH2)4O-, -(CH2)5O-, -(CH2)6O-, -(CH2)7O-, -(CH2)8O-, -(CH2)9O-, -(CH2)10O-, -CH2OCH2-, -CH2CH2O-, -(CH2CH2O)2-, -(CH2CH2O)3-, -(CH2CH2O)4-, -(CH2CH2O)5-, -CH2NH-, -CH2NHCH2-, -CH2CH2NH-, -CH2S-, -CH2SCH2-, and -CH2CH2S-. Examples of substituted heteroalkyl radicals include, but are not limited to, -C(O)CH2O-, -C(O)(CH2)2O-, -C(O)(CH2)3O-, -C(O)(CH2)4O-, -C(O)(CH2)5O-, -C(O)(CH2)6O-, -C(O)(CH2)7O-, -C(O)(CH2)8O-, -C(O)(CH2)9O-, -C(O)(CH2)10O-, -C(O)CH2OCH2CH2O-, -C(O)CH2O(CH2CH2O)2-, -C(O)CH2O(CH2CH2O)3-, -C(O)CH2O(CH2CH2O)4-, -C(O)CH2O(CH2CH2O)5-, -CH2NHC(O)CH2-, or -CH2CH2C(O)NH-.

[0184] The term "alkenyl" refers to linear or branched monovalent hydrocarbon radicals, which in one embodiment contain one, two, three, or four, and in another embodiment, one carbon-carbon double bond. Alkenyl groups are optionally substituted with one or more substituents Q described herein. The term "alkenyl" includes groups having "cis" or "trans" configurations or mixtures thereof, or alternatively, as understood by one of ordinary skill in the art, having "Z" or "E" configurations or mixtures thereof. For example, C2-6 alkenyl refers to linear unsaturated monovalent hydrocarbon radicals of 2 to 6 carbon atoms or branched unsaturated monovalent hydrocarbon radicals of 3 to 6 carbon atoms. In certain embodiments, alkenyl is a linear monovalent hydrocarbon radical of 2 to 20 (C2-20), 2 to 15 (C2-15), 2 to 10 (C2-10), or 2 to 6 (C2-6) carbon atoms, or a branched monovalent hydrocarbon radical of 3 to 20 (C3-20), 3 to 15 (C3-15), 3 to 10 (C3-10), or 3 to 6 (C3-6) carbon atoms. Examples of alkenyl include, but are not limited to, ethenyl, propenyl (including all isomeric forms, e.g., propen-l-yl, propen-2-yl, and allyl), and butenyl (including all isomeric forms, e.g., buten-l-yl, buten-2-yl, buten-3-yl, and 2-buten-l-yl).

[0185] The terms "alkenylene" and "alkenediyl" are used interchangeably herein to refer to a linear or branched divalent hydrocarbon radical containing one or more one, two, three, or four, in one embodiment, carbon-carbon double bonds, in another embodiment. The alkenediyl group is optionally substituted with one or more substituents Q described herein. The term "alkenediyl" includes groups having "cis" or "trans" configurations or mixtures thereof, or alternatively, as understood by one of ordinary skill in the art, having "Z" or "E" configurations or mixtures thereof. For example, C2-6alkenediyl refers to a linear unsaturated divalent hydrocarbon radical of 2 to 6 carbon atoms or a branched unsaturated divalent hydrocarbon radical of 3 to 6 carbon atoms. In certain embodiments, the alkenediyl group is a linear divalent hydrocarbon radical of 2 to 30 (C2-30), 2 to 20 (C2-20), 2 to 15 (C2-15), 2 to 10 (C2-10), or 2 to 6 (C2-6) carbon atoms, or a branched divalent hydrocarbon radical of 3 to 30 (C3-30), 3 to 20 (C3-20), 3 to 15 (C3-15), 3 to 10 (C3-10), or 3 to 6 (C3-6) carbon atoms. Examples of the alkanediyl group include, but are not limited to, polymethylene (including all isomeric forms, e.g., ethene-1, 1 -diyl ethene-1,2-diyl), polypropylene glycol (including all isomeric forms, e.g., 1 -propene-1, 1 -diyl, 1 -propene-1,2-diyl, and 1 -propene-1,3-diyl), polybutylene glycol (including all isomeric forms, e.g., 1 -butene-1, 1 -diyl, 1 -butene-1,2-diyl, and 1 -butene-1,4-diyl), poly pentene (including all isomeric forms, e.g., 1 -pentene-1, 1 -diyl, 1 -pentene-1,2-diyl, and 1 -pentene-1,5-diyl), hexanediyl (including all isomeric forms, e.g., 1 -hexene-1, 1 -diyl, 1 -hexene-1,2-diyl, 1 -hexene-1,3-diyl, 1 -hexene-1,4-diyl, 1 -hexene-1,5-diyl, and 1 -hexene-1,6-diyl).

[0186] The terms "heteroalkenylene" and "heteroalkenediyl" are used interchangeably herein to refer to a linear or branched divalent hydrocarbon radical that, in one embodiment, contains one or more, and in another embodiment, one, two, three, or four carbon-carbon double bonds, and which contains one or more heteroatoms, each heteroatom being independently selected from O, s, and N in the carbon hydrocarbon chain. The heteroalkene is optionally substituted with one or more substituents Q described herein. The term "heteroalkene" includes groups having "cis" or "trans" configurations or mixtures thereof, or alternatively, "Z" or "E" configurations or mixtures thereof, as understood by one of ordinary skill in the art. For example, C2-6heteroalkene refers to a linear unsaturated divalent hydrocarbon radical of 2 to 6 carbon atoms or a branched unsaturated divalent hydrocarbon radical of 3 to 6 carbon atoms. In certain embodiments, the heteroalkenyl is a linear divalent hydrocarbon radical of 2 to 20 (C2-20), 2 to 15 (C2-15), 2 to 10 (C2-10), or 2 to 6 (C2-6) carbon atoms, or a branched divalent hydrocarbon radical of 3 to 20 (C3-20), 3 to 15 (C3-15), 3 to 10 (C3-10), or 3 to 6 (C3-6) carbon atoms. Examples of heteroalkenyl groups include, but are not limited to: -CH=CHO-, -CH=CHOCH2-, -CH=CHCH2O-, -CH=CHS-, -CH=CHSCH2-, -CH=CHCH2S-, or -CH=CHCH2NH-.

[0187] The term "alkynyl" refers to a linear or branched monovalent hydrocarbon radical that, in one embodiment, contains one or more one, two, three, or four, and in another embodiment, one carbon-carbon triple bond. Alkynyl groups do not contain carbon-carbon double bonds. The alkynyl group is optionally substituted with one or more substituents Q described herein. For example, C2-6alkynyl refers to a linear unsaturated monovalent hydrocarbon radical of 2 to 6 carbon atoms or a branched unsaturated monovalent hydrocarbon radical of 4 to 6 carbon atoms. In certain embodiments, the alkynyl is a linear monovalent hydrocarbon radical of 2 to 20 (C2-20), 2 to 15 (C2-15), 2 to 10 (C2-10), or 2 to 6 (C2-6) carbon atoms, or a branched monovalent hydrocarbon radical of 4 to 20 (C4-20), 4 to 15 (C4-15), 4 to 10 (C4-10), or 4 to 6 (C4-6) carbon atoms. Examples of alkynyl groups include, but are not limited to, ethyl (-≡CH), propyl (including all isomeric forms, e.g., 1-propyl (-≡CCH3) and propargyl (-CH2C≡CH)), butyl (including all isomeric forms, e.g., 1-butyl-1-yl and 2-butyl-1-yl), pentyl (including all isomeric forms, e.g., 1-pentyl-1-yl and 1-methyl-2-butyl-1-yl), and hexyl (including all isomeric forms, e.g., 1-hexyl-1-yl and 2-hexyl-1-yl).

[0188] The terms "alkenylene" and "alkendiyl" are used interchangeably herein to refer to a linear or branched divalent hydrocarbon radical that contains one or more one, two, three, or four, in one embodiment, carbon-carbon double bonds, and, in another embodiment, one carbon-carbon triple bond. The alkenediyl group does not contain carbon-carbon triple bonds. As described herein, the alkenediyl group is optionally substituted with one or more substituents Q. For example, C2-6alkenediyl refers to a linear unsaturated divalent hydrocarbon radical of 2 to 6 carbon atoms or a branched unsaturated divalent hydrocarbon radical of 4 to 6 carbon atoms. In certain embodiments, the alkyldiyl is a linear divalent hydrocarbon radical of 2 to 30 (C2-30), 2 to 20 (C2-20), 2 to 15 (C2-15), 2 to 10 (C2-10), or 2 to 6 (C2-6) carbon atoms, or a branched divalent hydrocarbon radical of 4 to 30 (C4-30), 4 to 20 (C4-20), 4 to 15 (C4-15), 4 to 10 (C4-10), or 4 to 6 (C4-6) carbon atoms. Examples of alkyldiyl groups include, but are not limited to, polymethylene, polypropyne (including all isomeric forms, e.g., 1 -propyl- 1,3-diyl 1 -propyl-3,3-diyl), polybutyne (including all isomeric forms, e.g., 1 -butene- 1,3-diyl, 1 -butene- 1,4-diyl, and 2-butyne- 1, 1 -diyl), polyamylene (including all isomeric forms, e.g., 1 -pentane- 1,3-diyl, 1 -pentane- 1,4-diyl and 2-pentyne- 1, 1 -diyl), and polyhexyne (including all isomeric forms, e.g., 1 -hexyne- 1,3-diyl, 1 -hexyne- 1,4-diyl, and 2-hexyne- 1 -diyl).

[0189] The terms "heteroalkenylene" and "heteroalkendiyl" are used interchangeably herein to refer to a linear or branched divalent hydrocarbon radical that contains one or more one, two, three, or four, in one embodiment, carbon-carbon double bonds, and, in another embodiment, one carbon-carbon triple bond, and contains one or more heteroatoms in its backbone, each heteroatom being independently selected from O, S, and N. The heteroalkenyl group is optionally substituted with one or more substituents Q described herein. For example, C2-6heteroalkenyl refers to a linear unsaturated divalent hydrocarbon radical of 2 to 6 carbon atoms or a branched unsaturated divalent hydrocarbon radical of 4 to 6 carbon atoms. In certain embodiments, the heteroalkenyl is a linear divalent hydrocarbon radical of 2 to 30 (C2-30), 2 to 20 (C2-20), 2 to 15 (C2-15), 2 to 10 (C2-10), or 2 to 6 (C2-6) carbon atoms, or a branched divalent hydrocarbon radical of 4 to 30 (C4-30), 4 to 20 (C4-20), 4 to 15 (C4-15), 4 to 10 (C4-10), or 4 to 6 (C4-6) carbon atoms. Examples of heteroalkyldiyl groups include, but are not limited to, -C=CCH2O-, -C=CCH2S-, or -C=CCH2NH-.

[0190] The term "cycloalkyl" refers to a cyclic monovalent hydrocarbon radical, which is optionally substituted with one or more substituents Q described herein. In one embodiment, the cycloalkyl is saturated or unsaturated but non-aromatic and / or bridged or unbridged and / or fused bicyclic. In certain embodiments, the cycloalkyl group has 3 to 20 (C3-20), 3 to 15 (C3-15), 3 to 10 (C3-10), or 3 to 7 (C3-7) carbon atoms. In one embodiment, the cycloalkyl is monocyclic. In another embodiment, the cycloalkyl is bicyclic. In yet another embodiment, the cycloalkyl is tricyclic. In another embodiment, the cycloalkyl is polycyclic. Examples of cycloalkyl groups include, but are not limited to: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptyl, bicyclo[l. l. l]pentyl, bicyclo[2. l. l]hexyl, bicyclo[2.2. l]heptyl, bicyclo[2.2.2]octyl, decalinyl, and adamantyl.

[0191] The terms "cycloalkyl" and "cycloalkanediyl" are used interchangeably herein to refer to a cyclic divalent hydrocarbon radical, which can be optionally substituted with one or more substituents Q described herein. In one embodiment, the cycloalkanediyl can be saturated or unsaturated but non-aromatic and / or bridged and / or unbridged and / or fused bicyclic. In certain embodiments, the cycloalkanediyl has 3 to 30 (C3-30), 3 to 20 (C3-20), 3 to 15 (C3-15), 3 to 10 (C3-10), or 3 to 7 (C3-7) carbon atoms. Examples of polycycloalkane groups include, but are not limited to, polycyclopropane (including all isomeric forms, e.g., cyclopropane- 1, 1 -diyl, cyclopropane- 1,2-diyl), polycyclobutane (including all isomeric forms, e.g., cyclobutane- 1, 1 -diyl, cyclobutane- 1,2-diyl, and cyclobutane- 1,3-diyl), polycyclopentane (including all isomeric forms, e.g., cyclopentane- 1, 1 -diyl, cyclopentane- 1,2-diyl, and cyclopentane- 1,3-diyl), polycyclohexane (including all isomeric forms, e.g., cyclohexane- 1, 1 -diyl, cyclohexane- 1,2-diyl, cyclohexane- 1,3-diyl, cyclohexane- 1,4-diyl), cycloheptanediyl (including all isomeric forms, e.g., cycloheptane- 1, 1 -diyl, cycloheptane- 1,3-diyl, cycloheptane- 1,4-diyl), decanediyl (including all isomeric forms, e.g., decane- 1, 1 -diyl, decane- 1,2-diyl, and decane- 1,8-diyl), and adamantanediyl (including all isomeric forms, e.g., adamantane- 1,2-diyl, adamantane- 1,3-diyl, and adamantane- 1,8-diyl).

[0192] The term "aryl" refers to monovalent monocyclic and / or monovalent polycyclic arene radicals containing at least one aromatic carbon ring. In certain embodiments, aryl groups have from 6 to 20 (C6-20), from 6 to 15 (C6-15), or from 6 to 10 (C6-10) ring carbon atoms. Examples of aryl groups include, but are not limited to: phenyl, naphthyl, fluoronaphthyl, fluoronaphthyl, anthryl, phenanthryl, pyrenyl, biphenyl, and terphenyl. Aryl also refers to bicyclic or tricyclic carbon rings in which one ring is aromatic and the other rings can be saturated, partially unsaturated, or aromatic, such as indanyl, indenyl, or tetrahydronaphthyl (tetrafluoronaphthyl). In one embodiment, the aryl group is monocyclic. In another embodiment, the aryl group is bicyclic. In yet another embodiment, the aryl group is tricyclic. In another embodiment, the aryl group is polycyclic. In certain embodiments, the aryl group is optionally substituted with one or more substituents Q described herein.

[0193] The term "arylalkyl" or "arylalkyl" refers to monovalent alkyl groups substituted with one or more aryl groups. In certain embodiments, arylalkyl groups have from 7 to 30 (C7-30), from 7 to 20 (C7-20), or from 7 to 16 (C7-16) carbon atoms. Examples of arylalkyl groups include, but are not limited to: phenyl, phenethyl (including all isomeric forms, e.g., 1-phenylethyl and 2-phenylethyl), and phenylpropyl (including all isomeric forms, e.g., 1-phenylpropyl, 2-phenylpropyl, and 3-phenylpropyl). In certain embodiments, the arylalkyl group is optionally substituted with one or more substituents Q described herein.

[0194] The term "arylalkyl" or "arylalkyl" refers to monovalent alkyl groups substituted with one or more aryl groups. In certain embodiments, arylalkyl groups have from 7 to 30 (C7-30), from 7 to 20 (C7-20), or from 7 to 16 (C7-16) carbon atoms. Examples of arylalkyl groups include, but are not limited to: phenyl, phenethyl (including all isomeric forms, e.g., 1-phenylethyl and 2-phenylethyl), and phenylpropyl (including all isomeric forms, e.g., 1-phenylpropyl, 2-phenylpropyl, and 3-phenylpropyl). In certain embodiments, the arylalkyl group is optionally substituted with one or more substituents Q described herein.

[0195] The term "heteroaryl" refers to a monovalent monocyclic aromatic or a monovalent polycyclic aromatic containing at least one aromatic ring, wherein at least one aromatic ring contains one or more heteroatoms each independently selected from O, S, and N in the ring. For heteroaryl groups containing one heteroaromatic ring and one non-aromatic heterocyclic ring, the heteroaryl group is not bonded to the remainder of the molecule through its non-aromatic heterocyclic ring. Each ring of a heteroaryl group can contain one or two O atoms, one or two S atoms, and / or one to four N atoms; provided that the total number of heteroatoms in each ring is four or less, and each ring contains at least one carbon atom. In certain embodiments, a heteroaryl group has 5 to 20, 5 to 15, or 5 to 10 ring atoms. In one embodiment, a heteroaryl group is monocyclic. Examples of monocyclic heteroaryl groups include, but are not limited to: furanyl, imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyrimidinyl, pyridinyl, thiazolyl, thiazolyl, thiazolyl, tetrazolyl, triazinyl, and triazolyl. In another embodiment, a heteroaryl group is bicyclic.Examples of bicyclic heteroaryl groups include, but are not limited to: benzofuranyl, benzimidazolyl, benzothiazolyl, benzothiazolyl, benzothiazolyl, benzotriazolyl, benzoxazolyl, furopyridinyl (including all isomeric forms, e.g., furopyridinyl [2,3-b]pyridinyl, furopyridinyl [3,2-b]pyridinyl, furopyridinyl [3,2-c]pyridinyl, furopyridinyl [3,2-c]pyridinyl), imidazopyridinyl (including all isomeric forms, e.g., imidazopyridinyl [1,2-a]pyridinyl, imidazopyridinyl [4,5-b]pyridinyl, and imidazopyridinyl [4,5-c]pyridinyl), imidazothiazolyl (including all isomeric forms, e.g., imidazothiazolyl [2,1-b]thiazolyl and imidazothiazolyl [4,5-d]thiazolyl), indolizyl, indolyl, isobenzofuranyl, isobenzothiophenyl (i.e., benzo[c]thiophenyl), isoindolyl, isoquinolyl, naphthridinyl (including all isomeric forms, e.g., 1,5-naphthridinyl, 1,6-naphthridinyl, 1,7-naphthridinyl, and 1,8-naphthridinyl), oxazolopyridinyl (including all isomeric forms, e.g., oxazolopyridinyl [4,5-b]pyridinyl), oxazolopyridinyl [4,5-c]pyridinyl, oxazolopyridinyl [5,4-b]pyridinyl, oxazolopyridinyl [5,4-c]pyridinyl), phthalazinyl, pteridinyl, purinyl, pyrrolopyridinyl (including all isomeric forms, e.g., pyrrolopyridinyl [2,3-b]pyridinyl, pyrrolopyridinyl [2,3-c]pyridinyl, pyrrolopyridinyl [3,2-b]pyridinyl, pyrrolopyridinyl [3,2-c]pyridinyl), quinolinyl, quinolinyl, quinazolinyl, thiadiazolopyrimidinyl (including all isomeric forms, e.g., [1,2,5]thiadiazolopyrimidinyl [3,4-d]pyrimidinyl and [1,2,3]thiadiazolopyrimidinyl [4,5-d]pyrimidinyl)), thienopyrimidinyl (including all isomeric forms, e.g., [1,2,5]thiadiazolopyrimidinyl [3,5-d]pyrimidinyl), thienopyrimidinyl (including all isomeric forms, e.g., [1,2,5]thiadiazolopyrimidinyl), e.g., thienopyrimidinyl [2,3-b]pyridinyl, thienopyrimidinyl [2,3-c]pyridinyl, thienopyrimidinyl [3,2-b]pyridinyl, and thienopyrimidinyl [3,2-c]pyridinyl). In yet another embodiment, the heteroaryl group is tricyclic. Examples of tricyclic heteroaryl groups include, but are not limited to, acridinyl, benzindolyl, carbazolyl, dibenzofuranyl, o-benzofuranyl, phenanthrenediaminyl (including all isomeric forms).

[0196] The terms "heteroalkenyl" and "heteroalkenylene" are used interchangeably herein to refer to a divalent mono- or multicyclic hydrocarbon radical containing at least one alkenyl ring, wherein at least one ring contains one or more heteroatoms selected independently from O, S, and N. A heteroalkenyl group is not attached to the remainder of the molecule through a non-heteroatom of its ring. Each ring of a heteroalkenyl group can contain 1 or 2 O atoms, 1 or 2 S atoms, and / or 1 to 4 N atoms, provided that the total number of heteroatoms in each ring is 4 or less, and each ring contains at least 1 carbon atom. In certain embodiments, a heteroalkenyl group has 3 to 20, 3 to 15, or 3 to 10 ring atoms. Examples of monocyclic heteroalkenyl groups include, but are not limited to, 2-oxazolyl, 5-oxazolyl, 2-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-pyrrolyl, 3-pyrrolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 2-pyrazolyl, 3-pyrazolyl, 4-pyrazolyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl, 2-pyrimidinyl, 4-pyrimidinyl, 2-pyridazinyl, 3-pyridazinyl, 4-pyridazinyl, 2-triazolyl, 5-triazolyl, and 2-tetrazolyl.Examples of bicyclic heteroaryl groups include, but are not limited to: benzofurandiyl, benzimidazoli diyl, benzothiazol diyl, benzothiazol diyl, benzothiazol diyl, benzotriazol diyl, benzoxazol diyl, furanopyridindiyl (including all isomeric forms, e.g., furan[2,3-b]pyridindiyl, furan[3,2-b]pyridindiyl, furan[3,2-c]pyridindiyl, furan[3,4-c]pyridindiyl), imidazopyridindiyl (including all isomeric forms, e.g., imidazol[2,3-c]pyridindiyl, imidazol[3,4-b]pyridindiyl, imidazol[3,4-b]pyridindiyl), imidazopyridindiyl (including all isomeric forms, e.g., imidazol[1,2-a]pyridindiyl, imidazol[4,5-b]pyridindiyl, imidazol[4,5-c]-pyridindiyl), imidazothiazol diyl (including all isomeric forms, e.g., imidazol[2,1-b]thiazol diyl and imidazol[4,5-d]thiazol diyl), indolizindiyl, indolindiyl, isobenzofurandiyl, isobenzothiophendiyl (i.e., benzo[c]thiophendiyl), isoindolindiyl, isoquinolindiyl, naphthridindiyl (including all isomeric forms, e.g., 1,5-naphthridindiyl, 1,6-naphthridindiyl, 1,7-naphthridindiyl, and 1,8-naphthridindiyl); oxazolopyridindiyl (including all isomeric forms, e.g., oxazol[4,5-b]pyridindiyl, oxazol[4,5-c]pyridindiyl, oxazol[5,4-b]pyridindiyl, oxazol[5,4-b]pyridindiyl, pyridindiyl, pyrrolopyridindiyl, pyrrolopyridindiyl (including all isomeric forms, e.g., pyrrolo[2,3-b]pyridindiyl, pyrrolo[2,3-c]pyridindiyl, pyrrolo[3,2-b]-pyridindiyl, and pyrrolo[3,2-c]pyridindiyl), quinolindiyl, quinazolindiyl, thiadiazolopyrimidiyl (including all isomeric forms, e.g.,: [1,2,5]thiadiazol[3,4-d]-pyrimidiyl and [1,2,3]thiadiazol[4,5-d]pyrimidiyl) and thiophenopyridindiyl (including all isomeric forms, e.g., thiophen[2,3-b]pyridindiyl, thiophen[2,3-c]pyridindiyl, thiophen[3,2-b]pyridindiyl and thiophen[3,2-c]pyridindiyl). Examples of tricyclic heteroaryl groups include, but are not limited to, propellandiyl, benzindolindiyl, carbazolindiyl, dibenzofurandiyl, cyclonaphthendiyl, naphthendiyl (including all isomeric forms).

[0197] The term "heterocyclo" or "heterocycle" refers to a monovalent monocyclic non-aromatic ring system or a monovalent polycyclic ring system containing at least one non-aromatic ring, wherein one or more of the non-aromatic ring atoms is a heteroatom, each heteroatom is independently selected from O, S, and N; the remaining ring atoms are carbon atoms. For heterocyclyl groups containing heteroaromatic and non-aromatic heterocyclic rings, the heterocyclyl group is not bonded to the rest of the molecule through a heteroaromatic ring. In certain embodiments, a heterocyclyl group or heterocyclic group has 3 to 20, 3 to 15, 3 to 10, 3 to 8, 4 to 7, or 5 to 6 ring atoms. In certain embodiments, a heterocyclyl group is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which can be fused or bridged, and wherein a nitrogen atom or a sulfur atom can optionally be oxidized, a nitrogen atom can optionally be quaternized, and some rings can be partially or fully saturated, or aromatic. A heterocyclyl group can be attached at any heteroatom or carbon atom of the main structure, resulting in a stable compound. Examples of heterocyclyl acyl and heterocyclic groups include, but are not limited to: azepinyl, benzodioxo acyl, benzodithiinyl, chromioyl, decahydroisoquinoline acyl, dihydrobenzoisothiazole acyl, dihydrobenzoisothiazole acyl, dihydrobenzo[d][l,3]oxazinyl, 3,4-dihydrobenzo[c][l,2]oxazinyl, and 3,4-dihydrobenzo[d][l,2]oxazinyl), dihydrobenzothiophene acyl, dihydrobenzo[c]thiophene acyl, dihydrofuran acyl, dihydrobenzo[c]thiophene acyl, dihydrofuran acyl, dihydrofuran acyl, dihydroisoindole acyl, dihydropyranyl, dihydropyranyl, dihydropyranyl, dihydropyranyl, dihydropyranyl, dihydropyranyl, 1,4-dithianyl, furan acyl, imidazole acyl, imidazole acyl, indole acyl, iso-chromioyl, isothiazole acyl, isoxazole acyl, morpholinyl, octahydroindole acyl, octahydroisoindole acyl, oxazole acyl, oxazole acyl, oxo acyl, piperazinyl, piperidinyl, piperidinyl, 4-piperidinyl, pyrazole acyl, pyridine acyl, pyridine acyl, pyridine acyl, pyridine acyl, tetrahydrofuran acyl, tetrahydroisoquinoline acyl, tetrahydropyranyl, tetrahydrothiophenyl, thiazolidinyl, thiachromioyl, tetrahydroquinoline, and 1,3,5-trithianyl.

[0198] The term "heterocycloalkenyl" refers to a divalent monocyclic non-aromatic ring system or a divalent polycyclic ring system containing at least one non-aromatic ring, wherein one or more of the non-aromatic ring atoms are heteroatoms independently selected from O, S, and N; the remaining ring atoms are carbon atoms. For heterocyclyl groups containing both heteroaromatic and non-aromatic heterocyclic rings, the heterocyclyl group is bonded to the rest of the molecule through its non-aromatic heterocyclic ring. In certain embodiments, a heterocyclyl group has 3 to 20, 3 to 15, 3 to 10, 3 to 8, 4 to 7, or 5 to 6 ring atoms. In certain embodiments, a heterocycloalkenyl is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which can be fused or bridged, and wherein a nitrogen or sulfur atom can optionally be oxidized, a nitrogen atom can optionally be quaternized, and some rings can be partially or fully saturated, or aromatic. A heterocycloalkenyl can be attached to the main structure at any heteroatom or carbon atom, resulting in a stable compound. Examples of such heterocyclyl groups include, but are not limited to: azepindiyl, benzodiazepindiyl, benzodiazepindiyl, chromindiyl, decahydroisoquinolindiyl, dihydrobenzodiazepindiyl, dihydrobenzodiazepindiyl, dihydrobenzodiazepindiyl, 3,4-dihydrobenzo[d][l,3]oxazepindiyl, 3,4-dihydrobenzo[c][l,2]oxazepindiyl, 3,4-dihydrobenzo[d][l,2]oxazepindiyl, dihydrobenzodiazepindiyl, dihydrobenzo[c]oxazepindiyl, dihydrobenzodiazepindiyl, dihydrobenzodiazepindiyl, dihydrobenzodiazepindiyl, dihydrobenzodiazepindiyl, dihydrobenzodiazepindiyl, dihydroazepindiyl, dihydroazepindiyl, dihydroazepindiyl, dihydroazepindiyl, dihydroazepindiyl, dihydroazepindiyl, dihydroazepindiyl, dihydroisoindolindiyl, dihydropyridindiyl, dihydropyridindiyl, dihydropyridindiyl, dihydropyridindiyl, dioxazolindiyl, 1,4-dithiindiyl, furandiyl, imidazolidiyl, imidazolidiyl, imidazolidiyl, indolindiyl, isochlorindiyl, isothiazolidiyl, isoxazolidiyl, morpholindiyl, octahydroindolindiyl, octahydroisoindolindiyl, oxazolidiyl, oxazolidiyl, piperazindiyl, piperidindiyl, 4-piperidindiyl, pyrazolidiyl, pyrazolidiyl, pyrrolidiyl, pyrrolidiyl, quinolindiyl, tetrahydrofurandiyl, tetrahydroisoquinolindiyl, tetrahydropyrandiyl, tetrahydrothiophendiyl, thiazolidiyl, thiachromindiyl, tetrahydroquinolindiyl, and 1,3,5-trithiindiyl.

[0199] The Chinese name of DMSO is dimethyl sulfoxide;

[0200] The Chinese name of DMF is N,N-dimethylformamide;

[0201] The Chinese name of HOBt is 1-hydroxybenzotriazole;

[0202] The Chinese name of HBTU is O-benzotriazole-tetramethyluronium hexafluorophosphate;

[0203] DIPEA (DIEA) is N,N-diisopropylethylamine in Chinese;

[0204] DCM is dichloromethane in Chinese;

[0205] DMAP is 4-dimethylaminopyridine in Chinese;

[0206] DMT-Cl is 4,4'-dimethoxytritylchloride in Chinese;

[0207] MeOH is methanol in Chinese;

[0208] TBTU is O-benzotriazol-N,N,N',N'-tetramethyluronium tetrafluoroborate in Chinese;

[0209] The name of is solid phase carrier, such as macroporous aminomethyl resin (Resin);

[0210] SATA is S-acetylthioacetate N-succinimidyl-S-acetylthioacetate, which can be referred to Duncan, R.J., et al. 1983. Anal. Biochem.. 132, 68;

[0211] endo-BCN-NHS carbonate is (1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl methyl succinimidyl carbonate, which can be referred to Angew. Chem. Int. Ed. 2021, 60, 25905-25913;

[0212] N3-C5 NHS ester is azido-C5-succinimidyl ester;

[0213] Trt: trityl trityl

[0214] THF: tetrahydrofuran, English full name Tetrahydrofuran;

[0215] Pd / C: palladium-carbon catalyst;

[0216] DBU: bicyclic amidine, chemical name 1,8-diazabicyclo(5,4,0)-7-undecene;

[0217] Hexadecylamine: hexadecylamine;

[0218] Succinic Acid Monobenzyl Ester: succinic acid monobenzyl ester;

[0219] TFAPFP: pentafluorophenyl trifluoroacetate

[0220] Succinic Anhydride: succinic anhydride

[0221] Resin: resin;

[0222] NEt3: triethylamine;

[0223] t-Butyl Acrylate: tert-butyl acrylate

[0224] Hexadecylamine: hexadecylamine;

[0225] TFA: trifluoroacetic acid;

[0226] Suberic Acid Monobenzyl Ester: suberic acid monobenzyl ester.

[0227] Kylo-04-xdl-xiners-c5:

[0228] Cbz-Glu-OH: N-benzyloxycarbonyl-L-glutamic acid-5-methyl ester;

[0229] Azido-Aca-NHS: azido-C5-succinimidyl ester;

[0230] Tetrazine-NHS ester: tetrazine-succinimidyl ester.

[0231] Kylo-04-xdl-xiners-c5: is self-made, the structure is shown in the figure, and the rest of the above raw materials are from commercial purchase.

[0232] An RNA inhibitor consists of an oligonucleotide, a delivery ligand, an orthogonal reactive group, and a linker; the delivery ligand is coupled to the oligonucleotide through the orthogonal reactive group and the linker.

[0233] First part: delivery ligand

[0234] GalNAc ligand specifically binding to ASGPR receptor:

[0235] 1.11 GalNAc ligand 3'MVIP09 used in the following experiments:

[0236] The structure is shown as follows:

[0237] GalNAc ligand synthesis as disclosed in CN2023106555633, the synthesis process of 3'MVIP is introduced in the present application.

[0238] 1.12The structure of GalNAc ligand is not limited to this, and the structure of GalNAc ligand in CN2023106555633 and PCTCN2023095768 is introduced in the present application.

[0239] 1.2The structure of the auxiliary membrane permeable molecule ligand for assisting the membrane permeable oligonucleotide:

[0240] 1.2.1The structure of the auxiliary membrane permeable molecule ligand used in the following experiment is as follows:

[0241] The coupling mode of the auxiliary membrane permeable molecule ligand structure is not limited to this, the auxiliary membrane permeable molecule ligand can be connected to the end or inside of the nucleotide chain, and can be connected to the linker chemical structure through the connecting chain, and the connection position is not limited. The end of the nucleotide includes: the 3' end, 5' end of the positive strand, or the 3' end, 5' end of the negative strand; the inside of the nucleotide refers to the conjugation with the inside nucleotide, as an embodiment, the conjugation site can be the 3' position of the ribose five-membered ring of one of the nucleotides in the nucleotide chain, which can be conjugated with one or multiple, which is not limited, and the position of the nucleotide in the nucleotide chain is also not limited, which can be the 6th from the 5' end, or other positions.

[0242] The following is a schematic diagram of the auxiliary membrane permeable molecule ligand connected to the inside of the nucleotide chain:

[0243] The structure of the auxiliary membrane permeable molecule ligand is not limited to this, and the lipophilic compound can be coupled to the nucleotide chain or the linker chemical structure through the above coupling mode by a linker containing acyl, sulfide, disulfide, oxygen, PEG, alkyl and other functional groups. The linker can be single-chain, double-chain, triple-chain or quadruple-chain, and the structure of the linker is not limited.

[0244] Examples of lipophilic compounds include: Here, it is not exhaustive, and lipophilicity is a tendency of a compound to preferentially distribute in a non-polar lipid medium rather than in an aqueous phase. Any compound that preferentially distributes in a non-polar lipid medium can be applied to the present application, and the lipophilic compound can also be a lipophilic natural substance, such as vitamin E, saponin, cholesterol.

[0245] 1.2.2Synthesis process of auxiliary membrane permeable molecule ligand structure:

[0246] Example 1-1, 5' end loading of a single hexadecyl group

[0247] Ethanolamine was used as the starting material, the amino group was protected by Trt, and the hydroxyl group was subjected to Michael addition with methyl acrylate. The methyl ester was hydrolyzed to obtain a cross structure. The acid was condensed with 1-aminohexadecane, and the Trt protecting group was removed by trifluoroacetic acid. The naked amino group was condensed with octanedioic acid monomethyl ester. The methyl ester was hydrolyzed under alkaline conditions to prepare its activated ester, which was condensed with an oligonucleotide connected with a M2 monomer to be loaded onto the 5' end of the oligonucleotide.

[0248] Preparation of Yic-hd-c1: Ethanolamine (25.9) was dissolved in dichloromethane (300 mL) with triethylamine (53.63 g), and triphenylmethyl chloride (59.1 g) was added in batches. Stirring was performed at room temperature for 4 h. The reaction was quenched by adding water (800 mL), and the aqueous phase was extracted with dichloromethane (200 mL) once. The organic phases were combined, washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain a crude product of 71.2 g. The crude product was slurried with ethyl acetate and petroleum ether to obtain the product Yic-hd-c1 of 50.4 g with a yield of 78.3%.

[0249] Preparation of Yic-hd-c2: Yic-hd-c1 (20 g) and tetrabutylammonium bromide (2.13 g) were added to a two-phase system of dichloromethane (100 mL) and 33.3% sodium hydroxide solution (39.6 g), and tert-butyl acrylate (42.25 g) was added dropwise. The dropwise addition was completed in 20 min, and the reaction was performed at room temperature overnight. The aqueous phase was extracted with dichloromethane (50 mL) once, and the organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, rotary evaporated, and dried by oil pump to obtain a crude product of 28.93 g. Silica gel column purification was performed to obtain the product Yic-hd-c2 (27.97 g) with a yield of 98.3%.

[0250] Preparation of Yic-hd-c3: Yic-hd-c3 (0.43 g) was dissolved in a solution of methanol (14 mL) and THF (7 mL), 10 w / w% potassium hydroxide solution (3.08 g) was added, the reaction was heated at 60°C for 3 h, and then cooled to room temperature. After concentration, dichloromethane (20 mL) and water (10 mL) were added, and 5% acetic acid solution was added dropwise to adjust the pH to 6. The aqueous phase was extracted with dichloromethane (20 mL*2), and then the organic phases were combined and washed with saturated brine (30 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and then purified by silica gel column to obtain Yic-hd-c3 (0.38 g) with a yield of 84.0%.

[0251] Preparation of Yic-hd-c4: Yic-hd-c3 (1.20 g) was dissolved in dichloromethane (40 mL), TBTU (1.54 g) and DIPEA (1.65 g) were added, and the mixture was stirred at room temperature for 15 min, then hexadecylamine was added dropwise, and the mixture was stirred overnight. Water (50 mL) was added to quench the reaction, and the aqueous phase was extracted with dichloromethane (50 mL*3). The combined organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The crude product was purified by silica gel column chromatography to give Yic-hd-c4 (0.90 g) in a yield of 47.0%.

[0252] Preparation of Yic-hd-c5: Yic-hd-c4 (4.0 g) was dissolved in dichloromethane (120 mL), trifluoroacetic acid (25 mL) was added at room temperature, and the mixture was stirred for 4 h. The pH of the reaction solution was adjusted to 8 by dropwise addition of a protected sodium bicarbonate solution. The precipitate was filtered, and the filter cake was washed with water and dichloromethane, respectively. The filter cake was slurried in acetonitrile and then filtered. The filter cake was dried under oil pump vacuum to give Yic-hd-c5 (1.60 g), which was directly used in the next step.

[0253] Preparation of Yic-hd-c6: DMF (150 mL), monobenzyl octanedioate (1.4 g), TBTU (2.2 g), and DIPEA (5.0 mL) were added to a bottle containing Yic-hd-c5 (1.60 g). The reaction solution was stirred at room temperature overnight. The reaction solution was clear. The reaction was completed, and the DMF was removed by concentration under reduced pressure. The residue was dissolved in dichloromethane (200 mL), and the organic phase was washed with saturated sodium bicarbonate (100 mL), 10% citric acid (100 mL), and saturated brine (100 mL), respectively, and dried over anhydrous sodium sulfate. The filtrate was concentrated under reduced pressure, and the residue was slurried in methanol to give Yic-hd-c6 (2.13 g).

[0254] Yic-hd-c7: Yic-hd-c6 (2.1 g) was dissolved in dichloromethane (40 mL) and THF (80 mL). After stirring until complete dissolution, 10% Pd / C (0.3 g) was added, and the mixture was replaced with nitrogen for 3 times and replaced with hydrogen for 3 times. The mixture was hydrogenated at room temperature under normal pressure for 4 h. A large amount of solid was precipitated. The mixture was filtered with the aid of diatomite to remove the palladium catalyst. The filter cake was dissolved in trifluoroacetic acid, combined with the filtrate, and concentrated under reduced pressure. The residue was slurried in acetonitrile (150 mL) and then filtered. The filter cake was naturally air-dried to give Yic-hd-c7 (1.6 g).

[0255] 5'-HJYNSdp01: Yic-hd-c7 (0.60 g) was weighed and suspended in dichloromethane (30 mL), DIPEA (1.0 mL) was added, stirred at room temperature, then pentafluorophenyl trifluoroacetate (0.98 g) was added, after 2 h of reaction, the solvent was removed by rotary evaporation. The residue was slurried with water (20 mL) and acetonitrile (20 mL) once, and the obtained solid was filtered and dried by oil pump to obtain the product 3'-HJYNSdp01 (0.71 g).

[0256] Example 1-2: 3' end loaded mono-hexadecyl

[0257] 3'-HJYNSdp01 preparation: the intermediate of Trt deprotection group was condensed with octanedioic acid (this intermediate was prepared in the laboratory), the hydroxyl group was added with succinic anhydride to obtain a carboxylic acid, which was loaded onto the resin and used to synthesize 3' end loaded hexadecyl oligonucleotide.

[0258] Yic-hd-c5, 3'-HJYNSdp01-c1 preparation: Yic-hd-c5 crude (1.0 g) was weighed and dissolved in DMF (30 mL), Kylo-04-xdl-xiners-c5 (2.0 g) was added, TBTU (1.4 g) and DIPEA (1.5 g) were added, and the reaction was carried out at 50°C overnight. After the reaction was completed, the reaction solution was cooled to room temperature and poured into water (700 mL), stirred for 15 minutes, then filtered, and white solid was obtained. Purification on silica gel column to obtain 3'-HJYNSdp01-c1 (1.20 g), yield 81.3%. MS: 887.6024, found [M-H] 886.5986.

[0259] 3'-HJYNSdp01-c2 preparation: 3'-HJYNSdp01-c1 (2.42 g) was dissolved in dichloromethane (50 mL), DBU (1.25 g) and succinic anhydride (0.82 g) were added in turn, the reaction was carried out at room temperature for 1 h, then water (20 mL) was added to quench the reaction, the aqueous phase was extracted with dichloromethane (50 mL*3), the organic phase was combined and washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, rotary evaporated, and purified on a silica gel column to obtain the product 3'-HJYNSdp01-c2 (2.30 g), yield 85.5%. MS: MW 987.6184, found [M-H] 986.5987.

[0260] 3'-HJYNSdp01-PS preparation: 3'-HJYNSdp01-c2 (0.494 g), HBTU (0.23 g) and DMF (40 mL) were added into a 100 mL single-neck flask under nitrogen atmosphere. After 5 min of reaction at room temperature, DIPEA (0.348 mL) and aminomethyl resin (2.66 g) were added. The reaction was heated to 50 °C and reacted for 24 h. After cooling to room temperature, the solvent was removed by filtration and then washed with DMF (40 mL*3). The residual solid was capped, and after 3 h of reaction, it was filtered. The resin was washed with methanol and dichloromethane (20 mL*3), respectively. After drying, 3'-HJYNSdp01-PS resin (3.01 g) was obtained, and the degree of substitution was measured to be 141 μmol / g.

[0261] Examples 1-3: Bifunctional glutamic acids:

[0262] Cbz-Glu-OH was condensed with 1-amino hexadecane to give an amide, which was deprotected by hydrogenation to give a carboxylic acid. The pfp activated ester was prepared. After coupling, an oligonucleotide loaded with two hexadecyl groups was obtained.

[0263] Preparation of Erc-Glu-c1: Cbz-Glu-OH (20 g) and DIPEA (25.0 mL) were weighed and dissolved in dichloromethane (400 mL). Then TBTU (50.3 g) and hexadecylamine (36.0 g) were added, and the reaction was carried out at room temperature overnight. After the reaction was completed, the reaction solution was poured into mechanically stirred water (2.0 L), stirred for 30 min, and then filtered. The solid was successively slurried in water (2.0 L) and acetonitrile (500 mL), filtered, and dried to obtain the product Erc-glu-c1 (50.0 g). MS: MW 727.6227, found [M-H] 726.6193.

[0264] Preparation of Erc-Glu-c2: Erc-Glu-c1 (10.0 g) was weighed and added to ethanol (70 mL) and reacted at 60 °C. After the solution was clear, 10% Pd / C (0.9 g) was added, and the reaction was carried out under nitrogen for 3 times and then under hydrogen for 3 times. The hydrogenation reaction was carried out at room temperature for 3.5 h. After the reaction was completed, the palladium carbon was filtered with diatomite, and the ethanol was removed by rotary evaporation. After stirring in n-heptane (100 mL) for 30 min, the product Erc-Glu-c2 (6.5 g) was obtained by filtration and dried naturally. MS: MW 593.5859, found [M-H] 592.5806.

[0265] Preparation of Erc-glu-c3: Monobenzyl octanedioate (3.7 g) was dissolved in dichloromethane (800 mL), TBTU (5.4 g) and DIPEA (12.0 mL) were added, and the mixture was stirred at room temperature for 10 min. Then Erc-glu-c2 (6.90 g) was added, and DIPEA (8.0 mL) was added. The mixture was stirred at 40 °C overnight. After the reaction was completed, the dichloromethane was removed by concentration, and the residue was washed with water (200 mL), saturated sodium bicarbonate (200 mL), and acetonitrile (200 mL) in sequence. The solid was obtained by filtration and air-dried to obtain the product Erc-glu-c3 (9.0 g).

[0266] Preparation of Erc-glu-c4: Erc-glu-c3 (9.0 g) was weighed and dissolved in ethanol (100 mL) and reacted at 60 °C. After the solution was clear, 10% Pd / C (0.9 g) was added, and the mixture was replaced with nitrogen for 3 times and then replaced with hydrogen for 3 times. The hydrogenation reaction was carried out at room temperature for 8 h. After the reaction was completed, the mixture was filtered with diatomite and palladium carbon, and the ethanol was removed by rotary evaporation. The residue was washed with water (50 mL), dichloromethane (50 mL), methanol (50 mL), and acetonitrile (50 mL) in sequence. The solid was obtained by filtration and air-dried to obtain the product Erc-glu-c4 (7.5 g).

[0267] Preparation of 5'-HJYNSdp02: Erc-glu-c4 (2.00 g) was weighed and suspended in dichloroethane (300 mL). DIPEA (5.00 mL) was added, and the mixture was stirred at room temperature. Then pentafluorophenyl trifluoroacetate (1.60 g) was added, and the mixture was stirred overnight. The solvent was removed by rotary evaporation. The residue was washed with water (50 mL), saturated sodium bicarbonate (50 mL), and acetonitrile (50 mL) in sequence. The solid was obtained by filtration and air-dried to obtain the product 5'-HJYNSdp02 (2.0 g).

[0268] Examples 1-4: Dimeric glutamic acid resins

[0269] The condensation of bis (hexadecylamine) glutamic acid with Kylo-04-xdl-xiners-c5, the connection of the hydroxyl group with succinic anhydride, and the loading onto the resin can be used to prepare 3' loaded dimeric hexadecyl oligonucleotide.

[0270] Kylo-Erc-glu-c3 preparation: Kylo-04-xdl-xiners-c5 (2.25 g), TBTU (1.52 g) and Erc-glu-c2 (1.25 g) were weighed in a flask, dissolved in DMF (100 mL), added DIPEA (1.67 g), and reacted at 50°C overnight. After the reaction was completed, the reaction solution was directly poured into water (200 mL). Dichloromethane (200 mL*3) was extracted, the organic phase was combined, washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered, and rotary evaporated. Ethyl acetate (100 mL) was added to the crude product and heated to 50°C, maintained for half an hour, and then filtered while hot to obtain the product Kylo-Erc-Glu-c3 (1.9 g). MS: MW 1124.8480, MS found [M-H] 1123.8386.

[0271] Kylo-Erc-glu-c4 preparation: Kylo-Erc-glu-c3 (0.56 g) was weighed, dichloromethane (10 mL) was added, and then DBU (0.23 g) and succinic anhydride (0.15 g) were added, and the reaction was carried out at room temperature overnight. The reaction was quenched by adding water (50 mL), dichloromethane (50 mL*3) was extracted, the organic phase was combined, washed with saturated brine (100 mL), and then dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain a light yellow solid Kylo-Erc-glu-c4 (0.47 g). MS: [MW] 1224.8641, MS found 1223.8574.

[0272] 3'-HJYNSdp02-PS preparation: Under a nitrogen atmosphere, Kylo-Erc-glu-c4 (0.613 g), HBTU (0.23 g), and DMF (40 mL) were added to a 100 mL single-neck flask, and the reaction was carried out at room temperature for 5 minutes. DIPEA (0.48 mL) and aminomethyl resin (2.13 g) were added, heated to 50°C, and reacted for 24 hours. After cooling to room temperature, the solvent was removed by filtration, and then washed with DMF (40 mL*3). The residual solid was capped, reacted for 3 hours, and then filtered. The resin was washed with methanol and dichloromethane (20 mL*3), respectively, and then dried to obtain the resin loaded with 3'-HJYNSdp01-c2 (2.53 g), with a measured degree of substitution of 149 μmol / g.

[0273] Second part: oligonucleotide design and synthesis:

[0274] 2.1 siRNA design

[0275] A set of siRNAs targeting the human CFB gene (human: NCBI refseq ID GenBank NM_001710.5) was screened using a self-designed sequence screening software:

[0276] A set of siRNAs targeting the human C3 gene (human: NCBI refseq ID GenBank NM_000064.4) was screened using in-house designed sequence screening software:

[0277] A set of siRNAs targeting the human C5 gene (human: NCBI refseq ID GenBank NM_001735.3) was screened using in-house designed sequence screening software:

[0278] A set of siRNAs targeting the human TAU (MAPT) gene (human: NCBI refseq ID GenBank NM_016841.5) was screened using in-house designed sequence screening software:

[0279] A set of siRNAs targeting the human APP gene (human: NCBI refseq ID GenBank NM_201414.3) was screened using in-house designed sequence screening software:

[0280] The sense and antisense strands of the modified sequences in the inhibitors are shown in Table 1.

[0281] siRNAs were synthesized and annealed using conventional methods known in the art.

[0282] In certain embodiments, wherein the antisense strand and the sense strand form a duplex structure (complementary region) that includes at least 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides.

[0283] In some screening embodiments, the sense and antisense strands of the RNAi agent are selected from the sequences in Table 1 or differ from each sequence in Table 1 by one, two, or three nucleotides.

[0284] In some embodiments, the base pair complementarity of the sense strand in Table 1 and the corresponding antisense strand in Table 1 forms a dsRNA, which can be partially complementary or fully complementary. The partial complementarity can be at least 85% base pairing.

[0285] In some embodiments, the sense and antisense strands are not limited to the double-stranded combinations in Table 1, and any one of the sense strands in Table 1 can be base pair complementarily paired with any one of the antisense strands.

[0286] The present application aims to protect the core sequence of the sequences in Table 1, which is any stretch of at least 15 consecutive nucleotides of the above sequences, wherein at least 15 means 15, 16, 17, 18, 19, 20, 21, 22, 23, etc. In some embodiments, the sense strand is of formula (1): 5'-X core sequence Y-3', and the antisense strand is at least 85% complementary to the sense strand, X, Y comprising 0, 1, 2, 3, 4, 5, 6 nucleotides, and 0, 1, 2, 3 unpaired bases at the end position in the duplex are tolerated.

[0287] The antisense strand comprises consecutive nucleotides differing by 0, 1, 2, or 3 nucleotides from formula (2): 5'-X' core sequence Y'-3', and the antisense strand is at least 85% complementary to the sense strand, X', Y' comprising 0, 1, 2, 3, 4, 5, 6 nucleotides, and 0, 1, 2, 3 unpaired bases at the end position in the duplex are tolerated.

[0288] As an example, the core sequence allows for 0, 1, 2, 3 nucleotide differences, which can be base pairs according to the Watson-Crick principle or mismatches.

[0289] In some embodiments, the RNA inhibitors can be administered to cell lines for sequence screening by means of cell transfection or liposome-nucleic acid nanoparticle known to those skilled in the art. The methods for preparing lipids and liposome-nucleic acid nanoparticles are described in full in patents US9233971B2, US9080186B2, CN102985548B and CN103189057B, which are incorporated herein in their entirety.

[0290] In some embodiments, the amphipathic lipid in the lipid compound is preferably a macrocyclic lipid compound D1C1, T1C1, T1C6, T4C4, B2C1, B2C6, B2C7 and M10C1.

[0291] It is well known to those skilled in the art that dsRNAs having duplex structures of about 20 to 23 base pairs, e.g., 21 base pairs, have been shown to be particularly effective at inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer RNA duplexes are also effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). It can be reasonably expected that duplexes having one or both ends reduced or increased by a few nucleotides from a sequence in Tables 1, 2 can be similarly effective as compared to the dsRNAs described. Thus, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, 21, or more contiguous nucleotides derived from a sequence in Tables 1, 2 and differing in the ability to inhibit ANGPTL3 gene expression by no more than about 5, 10, 15, 20, 25, or 30% from a dsRNA comprising the full sequence are included within the scope of the application.

[0292] The dsRNAs described herein can further comprise one or more single-stranded nucleotide overhangs, e.g., 1, 2, 3, or 4 nucleotides. The nucleotide overhangs can comprise nucleotides / nucleoside analogs or combinations thereof, including deoxynucleotides. The overhangs can be on the sense strand, the antisense strand, or a combination thereof. In addition, the nucleotides of the overhangs can be present on the 5' end, the 3' end, or both ends of the antisense strand or the sense strand of the dsRNA. The overhangs can be formed by one strand being longer than the other strand, or by the two strands of the same length being staggered. When the overhang is on the antisense strand, it can form a mismatch or a complementarity with the ANGPTL3 mRNA or can be another sequence. For example, the overhang is on the 3' end of the sense strand, or alternatively, on the 3' end of the antisense strand.

[0293] The dsRNAs can also have blunt ends, by which is meant that there are no unpaired nucleotides, i.e., no nucleotide overhangs, at that end of the dsRNA. The blunt ends can be on the 5' end of the antisense strand and the 3' end of the sense strand, or vice versa, or double-blunt-ended, by which is meant that the dsRNA is double-stranded over its entire length, i.e., there are no nucleotide overhangs at either end of the molecule.

[0294] In some embodiments, the sense strand or the antisense strand of the dsRNA has a nucleotide overhang of 1, 2, 3, or 4 nucleotides at the 3' end, while the 5' end is blunt.

[0295] In some embodiments, the overhangs are present on both the 3' end of the sense strand and the 3' end of the antisense strand, and the overhangs comprise 1, 2, 3, or 4 nucleotides. The overhangs include, but are not limited to, TT, UU, AU, or UA.

[0296] In some embodiments, the dsRNA is a 19, 21 or 23 nucleotide long double- ended blunt-ended duplex, which is double-stranded over its entire length, i.e., there are no overhanging nucleotides at either end of the molecule.

[0297] In some embodiments, the dsRNA has a length of 21 nucleotides, and both the sense and antisense strands have 2 nucleotide overhangs at the 3' end.

[0298] To enhance the stability of the RNA inhibitors described herein in vivo, the sense and antisense strands of the RNA inhibitors can be modified without affecting or even enhancing their activity, wherein the nucleotides can have a modifying group, and the entire strand or part of the strand can be modified. In some embodiments, one or more nucleotides on the sense and / or antisense strand are modified to form a modified nucleotide.

[0299] In some embodiments, the sense and antisense strands of the RNA inhibitors (e.g., dsRNA) described herein are unmodified. In other embodiments, the sense and antisense strands of the RNA inhibitors described herein are chemically modified or conjugated as known in the art and described herein to enhance stability or other advantageous properties. In other embodiments of the application, all or substantially all of the nucleotides of the RNA inhibitors described herein can be modified, i.e., there are no more than 5, 4, 3, 2, or 1 unmodified nucleotides in the strand of the RNA inhibitor.

[0300] The sense and antisense strands of the RNA inhibitors described herein can be synthesized and / or modified using methods well known in the art, such as those described in "Current protocols in nucleic acid chemistry", Beaucage, S. L. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, incorporated herein by reference. In the RNA inhibitors provided herein, neither the sense nor the antisense strand of the RNA inhibitors need be uniformly modified, and one or more than one modification can be incorporated into individual nucleotides of the RNA inhibitors.

[0301] In some embodiments, the nucleotide modifications include: 5' terminal modified nucleotides, 3' terminal modified nucleotides, base modifications, sugar modifications or substitutions, backbone modifications. 5' terminal modifications refer to phosphorylation, conjugation, inverted linkages. 3' terminal modifications refer to conjugation, DNA nucleotides, inverted linkages, etc.; base modifications refer to substitutions with stabilizing bases, destabilizing bases, or base pairing with extended partner pools, removal of bases (abasic nucleotides), or conjugated bases. Sugar modifications are generally at the 2' position or 4' position. Backbone modifications refer to modifications or substitutions of phosphodiester linkages.

[0302] Specific nucleotide modifications can include, but are not limited to: 5' end phosphorous group containing nucleotide modifications, vinylphosphonate deoxyribonucleotides, vinylphosphonate containing nucleotides, and cyclopropylphosphonate containing nucleotides, 3' terminal deoxythymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxyl modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2-O-(N-methylacetamide) modified nucleotides, 3'-O-methoxy (2' internucleosidic linkage) nucleotides, 2'-F-arabino nucleotides, 5'-Me / 2'-fluoro nucleotides, locked nucleotides, unlocked nucleotides, unlocked nucleobase analogs, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, morpholino nucleotides, phosphoramidates, non-natural base containing nucleotides, tetrahydropyran modified nucleotides, 1,5- deoxyhexitol modified nucleotides, cyclohexenyl modified nucleotides, methylphosphonate group containing nucleotides, thermally unstable nucleotides, GNA, deoxyribonucleotides, nucleotide analogs, morpholino nucleotides, abasic nucleotides, 3' to 3' linked (inverted) nucleotides, bridged nucleotides, peptide nucleic acids (PNAs).

[0303] 5' end phosphorous group containing nucleotide modifications can be 5'-phosphate nucleotides or 5'-phosphate analog containing nucleotide deoxyribonucleotides; having including but not limited to: 5' end phosphate (5'-P), 5' end thiophosphate (5'-PS), 5-'end thiophosphodiester (5'-PS2), 5' end vinylphosphonate (5'-VP), 5' end methylphosphonate (MePhos), or 5'-deoxy-5'-C-malonate when the 5' end phosphorous group is a 5' end group vinylphosphate (5'-VP), the 5'-VP can be a 5'-E-VP isomer (i.e. trans vinylphosphate), a 5'-Z-VP isomer (i.e. cis vinylphosphate), or a mixture thereof.

[0304] Intersubunit modifications can include, but are not limited to: phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, alkylphosphonates, phosphinates, phosphoramidates, thiocarbonyl phosphoramidates, thiocarbonyl alkylphosphonates, thiocarbonyl alkylphosphotriesters, boranophosphates, also encompassing various salts, free acids.

[0305] Terminal modification of the sense strand or antisense strand can avoid exonuclease degradation, enhance nuclease stability, such as a cap structure: inverted deoxy abasic cap, abbreviated as invAb. invAb is well known in the art, and specific performance verification is described in F. Czauderna, Nucleic Acids Res., 2003, 31(11), 2705-16.

[0306] In some embodiments, the -OH of the 2' position of the sugar group of the nucleotide of the sense strand and / or the antisense strand can be deoxidized to -H, forming a 2'-deoxy modified nucleotide. Or dTdT is connected at the 3' end of the antisense strand to perform deoxy nucleotide terminal modification.

[0307] In some embodiments, there are at least two consecutive phosphorothioate bonds between the nucleotides of the sense strand and / or the antisense strand. Further, there are at least two consecutive phosphorothioate bonds between the 3 consecutive nucleotides of at least one end of the sense strand and / or the antisense strand.

[0308] In some embodiments, the sense strand and the antisense strand of the RNA inhibitor of the present application are selected from the following Table 1:

[0309] Table 1 sense strand and antisense strand of modified sequence in inhibitor

[0310] wherein G = 2'-O-methyl guanylic acid, A = 2'-O-methyl adenylic acid, U = 2'-O-methyl uridylic acid, C = 2'-O-methyl cytidylic acid; Gs = 2'-O-methyl-3'-thio guanylic acid, As = 2'-O-methyl-3'-thio adenylic acid, Us = 2'-O-methyl-3'-thio uridylic acid, Cs = 2'-O-methyl-3'-thio cytidylic acid; fG = 2'-fluoro guanylic acid, fA = 2'-fluoro adenylic acid, fU = 2'-fluoro uridylic acid, fC = 2'-fluoro cytidylic acid; fGs = 2'-fluoro-3'-thio guanylic acid, fAs = 2'-fluoro-3'-thio adenylic acid, fUs = 2'-fluoro-3'-thio uridylic acid, fCs = 2'-fluoro-3'-thio cytidylic acid, T = 2'-O-methyl deoxythymidylic acid, Ts = 2'-O-methyl-3'-thio deoxythymidylic acid, dA = 2'-deoxy adenylic acid, dT = 2'-deoxy thymidylic acid, dTs = 2'-deoxy-3'-thio thymidylic acid.

[0311] The delivery vector types and attachment positions of DDP04, DDP0401, DDP0402, DDP0403, DDP0404 and DDP0405 in Table 1 are shown in Table 2.

[0312] Table 2 Summary of delivery vectors for DDP04 family compounds

[0313] The antisense strands of siRNA 1 and siRNA 2 in DDP05, DDP0501 and DDP0502 in Table 1 are attached to delivery vector MVIP09 at their 3' ends, respectively, as described in the following examples.

[0314] The antisense strands of siRNA 1 and siRNA 2 in DDP06, DDP0601 and DDP0602 in Table 1 are attached to delivery vector MVIP09 at their 3' ends, respectively, as described in the following examples.

[0315] The antisense strands of siRNA 1 and siRNA 2 in DDP07, DDP0701 and DDP0702 in Table 1 are attached to delivery vector MVIP09 at their 3' ends, respectively, as described in the following examples.

[0316] The antisense strands of siRNA 1 and siRNA 2 in DDP08 in Table 1 are attached to delivery vector MVIP09 at their 3' ends, respectively, as described in the following examples.

[0317] The antisense strands of siRNA 1 and siRNA 2 in DDP09 in Table 1 are attached to delivery vector MVIP09 at their 3' ends, respectively, as described in the following examples.

[0318] The sense strands of siRNA 1 and siRNA 2 in DDP 10 in Table 1 were each linked to delivery vector MVIP09 at the 3' end, as described in the examples below.

[0319] The sense strands of siRNA 1 and siRNA 2 in DDP 11 in Table 1 were each linked to delivery vector MVIP09 at the 3' end, as described in the examples below.

[0320] The structures of the dual-target siRNA inhibitors in Table 1 are listed as follows:

[0321] DDP03-DS001:

[0322] siRNA 1: sense strand = 5' AsAsfGCfAAfGAfUfAfUUfUUfAUfAAsUsA 3';

[0323] antisense strand = 5' UsfAsfUUfAUAfAAfAAUAfUCfUUfGCUUsUsU dTdT 3';

[0324] siRNA 2: sense strand = 5' GsAsGCCGfUUfCfUfCUACAAUUAsCsU 3';

[0325] antisense strand = 5' AsfGsUAAfUUfGfUAGAGfAAfCGGCUCsGsG 3';

[0326] DDP02-DS002:

[0327] siRNA 1: sense strand = 5' UsUsGUCCfCAfGfUfAUUCUAUGUsUsU 3';

[0328] antisense strand = 5' AsfAsAfCAfUAfGAAUACfUGfGGACAAsCsG 3';

[0329] siRNA 2: sense strand = 5' GsGsUGUUfGAfCfAfGAUACAUsCsU 3';

[0330] antisense strand = 5' AsfGsAUGfUAfUCUGUCfAAfCACCsAsU 3';

[0331] DDP02-DS003:

[0332] siRNA 1: sense strand = 5' UsUsGUCCfCAfGfUfAUUCUAUGUsUsU 3';

[0333] Sense strand = 5' GsGUGUfGfAfCfAfGAUACAUsCsU 3';

[0334] siRNA2: Sense strand = 5' GsGsUGUUfGAfCfAfGAUACAUsCsU 3';

[0335] Antisense strand = 5' AsfGsAUGfUAfUCUGUCfAAfCACCsAsU 3';

[0336] DDP02-DS004:

[0337] siRNA1: Sense strand = 5' UsUsGUCCfCAfGfUfAUUCUAUGUsUsU 3';

[0338] Antisense strand = 5' AsfAsAfCAfUAfGAAUACfUGfGGACAAsCsG 3';

[0339] siRNA2: Sense strand = 5' GsGsUGUUfGAfCfAfGAUACAUsCsU 3';

[0340] Antisense strand = 5' AsdGsAUdGUdAUCUGTCfAACACCsAsU 3';

[0341] DDP02-DS005:

[0342] siRNA1: Sense strand = 5' UsUsGUCCfCAfGfUfAUUCUAUGUsUsU 3';

[0343] Antisense strand = 5' AsdAsACdAUdAGAAUdACfUGGGACAAsCsG 3';

[0344] siRNA2: Sense strand = 5' GsGsUGUUfGAfCfAfGAUACAUsCsU 3';

[0345] Antisense strand = 5' AsdGsAUdGUdAUCUGTCfAACACCsAsU 3';

[0346] DDP02-DS006:

[0347] siRNA1: Sense strand = 5' UsUsGUCCfCAfGfUfAUUCUAUGUsUsU 3';

[0348] Sense: 5' AsdAsACdAUdAGAAUdACfUGGGACAAsCsG 3';

[0349] siRNA2: Sense: 5' AsdAsACdAUdAGAAUdACfUGGGACAAsCsG 3';

[0350] Antisense: 5' UsdAsUAdGATGUAGTAfGAAUUUCUsCsU 3';

[0351] DDP02-DS007:

[0352] siRNA1: Sense: 5' AsdAsACdAUdAGAAUdACfUGGGACAAsCsG 3';

[0353] Antisense: 5' UsdAsUAdGATGUAGTAfGAAUUUCUsCsU 3';

[0354] siRNA2: Sense: 5' AsdAsACdAUdAGAAUdACfUGGGACAAsCsG 3';

[0355] Antisense: 5' UsdAsUAdGATGUAGTAfGAAUUUCUsCsU 3';

[0356] DDP02-DS008:

[0357] siRNA1: Sense: 5' AsdAsACdAUdAGAAUdACfUGGGACAAsCsG 3';

[0358] Antisense: 5' UsdAsUAdGATGUAGTAfGAAUUUCUsCsU 3';

[0359] siRNA2: Sense: 5' AsdAsACdAUdAGAAUdACfUGGGACAAsCsG 3';

[0360] Antisense: 5' UsdAsUAdGATGUAGTAfGAAUUUCUsCsU 3';

[0361] DDP02-DS009:

[0362] siRNA1: Sense: 5' AsdAsACdAUdAGAAUdACfUGGGACAAsCsG 3';

[0363] Sense: 5' AsdAsACdAUdAGAAUdACfUGGGACAAsCsG 3' Antisense: 5' UsfAsUGAfAGfCAAUUCfUCfCUCAGCsAsC 3'

[0364] siRNA2: Sense: 5' GsCsUGAGfGAfGfAfAUUGCUUCAsUsA 3' Antisense: 5' UsfAsUGAfAGfCAAUUCfUCfCUCAGCsAsC 3'

[0365] siRNA2: Sense: 5' GsCsUGAGfGAfGfAfAUUGCUUCAsUsA 3' Antisense: 5' UsfAsUGAfAGfCAAUUCfUCfCUCAGCsAsC 3'

[0366] DDP02-DS010:

[0367] siRNA2: Sense: 5' GsCsUGAGfGAfGfAfAUUGCUUCAsUsA 3' Antisense: 5' UsfAsUGAfAGfCAAUUCfUCfCUCAGCsAsC 3'

[0368] siRNA2: Sense: 5' GsCsUGAGfGAfGfAfAUUGCUUCAsUsA 3' Antisense: 5' UsfAsUGAfAGfCAAUUCfUCfCUCAGCsAsC 3'

[0369] siRNA2: Sense: 5' GsCsUGAGfGAfGfAfAUUGCUUCAsUsA 3' Antisense: 5' UsfAsUGAfAGfCAAUUCfUCfCUCAGCsAsC 3'

[0370] siRNA2: Sense: 5' GsCsUGAGfGAfGfAfAUUGCUUCAsUsA 3' Antisense: 5' UsfAsUGAfAGfCAAUUCfUCfCUCAGCsAsC 3'

[0371] DDP02-DS011:

[0372] siRNA2: Sense: 5' GsCsUGAGfGAfGfAfAUUGCUUCAsUsA 3' Antisense: 5' UsfAsUGAfAGfCAAUUCfUCfCUCAGCsAsC 3'

[0373] siRNA2: Sense: 5' GsCsUGAGfGAfGfAfAUUGCUUCAsUsA 3' Antisense: 5' UsfAsUGAfAGfCAAUUCfUCfCUCAGCsAsC 3'

[0374] siRNA2: Sense: 5' GsCsUGAGfGAfGfAfAUUGCUUCAsUsA 3' Antisense: 5' UsfAsUGAfAGfCAAUUCfUCfCUCAGCsAsC 3'

[0375] siRNA2: Sense: 5' GsCsUGAGfGAfGfAfAUUGCUUCAsUsA 3' Antisense: 5' UsfAsUGAfAGfCAAUUCfUCfCUCAGCsAsC 3'

[0376] DDP02-DS012:

[0377] siRNA2: Sense: 5' GsCsUGAGfGAfGfAfAUUGCUUCAsUsA 3' Antisense: 5' UsfAsUGAfAGfCAAUUCfUCfCUCAGCsAsC 3'

[0378] Antonym chain = 5'AsfAsAfCAfUAfGAAUACfUGfGGACAAsCsG 3';

[0379] siRNA2: positive strand = 5'AsGsAAAUfUCfUfAfCUACAUCUAsUsA 3';

[0380] Antonym chain = 5'UsdAsUAdGATGUAGTAfGAAUUUCUsCsU 3';

[0381] DDP04-DS001:

[0382] siRNA1: positive strand = 5'UsUsGUCCfCAfGfUfAUUCUAUGUsUsU3';

[0383] Antonym chain = 5'AsdAsACdAUdAGAAUdACfUGGGACAAsCsG3';

[0384] siRNA2: positive strand = 5'AsGsAAAUfUCfUfAfCUACAUCUAsUsA3';

[0385] Antonym chain = 5'UsdAsUAdGATGUAGTAfGAAUUUCUsCsU3';

[0386] DDP04-DS003:

[0387] siRNA1: positive strand = 5'CsUsAGACfCUfGUdTUUGCUUUUsGsU3';

[0388] Antonym chain = 5'AsfCsAfAfAfAGfCAfAAfACfAGfGUfCUAGsAsA3';

[0389] siRNA2: positive strand = 5'GsUsCAUCfCAfCfAfAUGAGAGUAsCsA3';

[0390] Antisense chain = 5'UsfGsUAC(Tgn)CUCAUUGfUGfGAUGACsGsA3';

[0391] DDP04-DS004:

[0392] siRNA1: positive strand = 5'CsUsAGACfCUfGUdTUUGCUUUUsGsU3';

[0393] Sense strand = 5' GsAsGCGACAfGfUfAUUCUCAGUsGsU 3';

[0394] siRNA2: Sense strand = 5' GsAsGCGACAfGfUfAUUCUCAGUsGsU 3';

[0395] Sense strand = 5' GsAsGCGACAfGfUfAUUCUCAGUsGsU 3';

[0396] DDP04-DS005:

[0397] siRNA1: Sense strand = 5' GsUsCAACAUfAfUfUUGAUCAGUsCsU 3';

[0398] Sense strand = 5' GsAsGCGACAfGfUfAUUCUCAGUsGsU 3';

[0399] siRNA2: Sense strand = 5' GsAsGCGACAfGfUfAUUCUCAGUsGsU 3';

[0400] Sense strand = 5' GsAsGCGACAfGfUfAUUCUCAGUsGsU 3';

[0401] DDP04-DS006:

[0402] siRNA1: Sense strand = 5' GsUsCAACAUfAfUfUUGAUCAGUsCsU 3';

[0403] Sense strand = 5' GsAsGCGACAfGfUfAUUCUCAGUsGsU 3';

[0404] siRNA2: Sense strand = 5' GsUsCAACAUfAfUfUUGAUCAGUsCsU 3';

[0405] Sense strand = 5' GsUsCAACAUfAfUfUUGAUCAGUsCsU 3';

[0406] DDP04-DS00601:

[0407] siRNA1: Sense strand = 5' GsUsCAACAUfAfUfUUGAUCAGUsCsU 3';

[0408] Antisense strand = 5' AsdGsACdTGdAUCAAdAUfAUGUUGAGsCsU 3';

[0409] siRNA2: Sense strand = 5' CsUsAGACfCUfGUdTUUGCUUUUsGsU 3';

[0410] Antisense strand = 5' AsfCsAfAfAfAGfCAfAAfACfAGfGUfCUAGsAsA 3';

[0411] DDP04-DS00703:

[0412] siRNA1: Sense strand = 5' AsGsAAAUfUCfUfAfCUACAUCUAsUsA 3';

[0413] Antisense strand = 5' UsdAsUAdGATGUAGTAfGAAUUUCUsCsU 3';

[0414] siRNA2: Sense strand = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0415] Antisense strand = 5' AsdAsAUTCdAGGAATUfCCUGCUUCsUsU 3';

[0416] DDP0401-DS00102:

[0417] siRNA1: Sense strand = 5' UsUsGUCCfCAfGfUfAUUCUAUGUsUsU 3';

[0418] Antisense strand = 5' AsdAsACdAUdAGAAUdACfUGGGACAAsCsG 3';

[0419] siRNA2: Sense strand = 5' AsGsAAAUfUCfUfAfCUACAUCUAsUsA 3';

[0420] Antisense strand = 5' VPUsdAsUAdGATGUAGTAfGAAUUUCUsCsU 3';

[0421] DDP0404-DS00702:

[0422] siRNA1: Sense strand = 5' GsCsUGAGfGAfGfAfAUUGCUUCAsUsA 3';

[0423] Sense strand = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0424] Sense strand = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0425] Sense strand = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0426] DDP0405-17 DS00102:

[0427] Sense strand = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0428] Sense strand = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0429] Sense strand = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0430] Sense strand = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0431] DDP0405-27 DS00702:

[0432] Sense strand = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0433] Sense strand = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0434] Sense strand = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0435] Sense strand = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0436] DDP0404- DS00601:

[0437] siRNA1 : sense strand = 5' CsUsCAACAUfAfUfUUGAUCAGUsCsU 3';

[0438] antisense strand = 5' AsdGsACdTGdAUCAAdAUfAUGUUGAGsCsUdTdT 3';

[0439] siRNA2: sense strand = 5' CsUsAGACfCUfGUdTUUGCUUUUsGsU 3';

[0440] antisense strand = 5' AsfCsAfAfAfAGfCAfAAfACfAGfGUfCUAGsAsAdTdT 3';

[0441] DDP0404-DS00503:

[0442] siRNA1 : sense strand = 5' CsUsAGACfCUfGUdTUUGCUUUUsGsU 3';

[0443] antisense strand = 5' AsfCsAfAfAfAGfCAfAAfACfAGfGUfCUAGsAsA 3';

[0444] siRNA2: sense strand = 5' CsAsGCUCCUfUfAfUUGUUAUACsGsA 3';

[0445] antisense strand = 5' VPUsfCsGfUAfUAACAAfUAfAGfGAfGCsfUsG 3';

[0446] DDP0404-DSS00503:

[0447] siRNA1 : sense strand = 5' CsUsAGACfCUfGUdTUUGCUUUUsGsUrS 3';

[0448] antisense strand = 5' AsfCsAfAfAfAGfCAfAAfACfAGfGUfCUAGsAsA 3';

[0449] siRNA2: sense strand = 5' CsAsGCUCCUfUfAfUUGUUAUACsGsArS 3';

[0450] antisense strand = 5' VPUsfCsGfUAfUAACAAfUAfAGfGAfGCsfUsG 3';

[0451] DDP0402-DS00704:

[0452] siRNA1: sense strand = 5' AsGsAAAUfUCfUfAfCUACAUCUAsUsA 3';

[0453] antisense strand = 5' UsdAsUAdGAdTGUAGdTAfGAAUUUCUsCsUdTdT 3';

[0454] siRNA2: sense strand = 5' GsUsCUAGfUCfAfAfCUUAAUUGAsGsA 3';

[0455] antisense strand = 5' UsdCsUCdAAdTUAAGdTUfGACUAGACsAsCdTdT 3';

[0456] DDP0404-DS004:

[0457] siRNA1: sense strand = 5' CsUsAGACfCUfGUdTUUGCUUUUsGsU 3';

[0458] antisense strand = 5' AsfCsAfAfAfAGfCAfAAfACfAGfGUfCUAGsAsAdTdT 3';

[0459] siRNA2: sense strand = 5' GsAsGCGACAfGfUfAUUCUCAGUsGsU 3';

[0460] antisense strand = 5' AsfCsAfCUfGAfGAAUACfUGfUCfGCsfUsCdTdT 3';

[0461] DDP0404-DSS004:

[0462] siRNA1: sense strand = 5' CsUsAGACfCUfGUdTUUGCUUUUsGsUrS 3';

[0463] antisense strand = 5' AsfCsAfAfAfAGfCAfAAfACfAGfGUfCUAGsAsAdTdT 3';

[0464] siRNA2: sense strand = 5' GsAsGCGACAfGfUfAUUCUCAGUsGsUrS 3';

[0465] antisense strand = 5' AsfCsAfCUfGAfGAAUACfUGfUCfGCsfUsCdTdT 3';

[0466] DDP0404-DS00501:

[0467] siRNA1: sense strand = 5' CsUsAGACfCUfGUdTUUGCUUUUsGsU 3';

[0468] antisense strand = 5' AsfCsAfAfAfAGfCAfAAfACfAGfGUfCUAGsAsAdTdT 3';

[0469] siRNA2: sense strand = 5' CsAsGCUCCUfUfAfUUGUUAUACsGsA 3';

[0470] antisense strand = 5' VPUsfCsGfUAfUAACAAfUAfAGfGAfGCsfUsGdTdT 3';

[0471] DDP0404-DSS00501:

[0472] siRNA1: sense strand = 5' CsUsAGACfCUfGUdTUUGCUUUUsGsUrS 3';

[0473] antisense strand = 5' AsfCsAfAfAfAGfCAfAAfACfAGfGUfCUAGsAsAdTdT 3';

[0474] siRNA2: sense strand = 5' CsAsGCUCCUfUfAfUUGUUAUACsGsArS 3';

[0475] antisense strand = 5' VPUsfCsGfUAfUAACAAfUAfAGfGAfGCsfUsGdTdT 3';

[0476] DDP0404-DS006:

[0477] siRNA1: sense strand = 5' CsUsAGACfCUfGUdTUUGCUUUUsGsU 3';

[0478] antisense strand = 5' AsfCsAfAfAfAGfCAfAAfACfAGfGUfCUAGsAsAdTdT 3';

[0479] siRNA2: sense strand = 5' CsUsCAACAUfAfUfUUGAUCAGUsCsU 3';

[0480] Sense = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0481] DDP0404-DS00704:

[0482] siRNA1: Sense = 5' GsCsUGAGfGAfGfAfAUUGCUUCAsUsA 3';

[0483] Antisense = 5' VPUsdCsUCdAAdTUAAGdTUfGACUAGACsAsCdTdT 3';

[0484] siRNA2: Sense = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0485] Antisense = 5' VPUsdCsUCdAAdTUAAGdTUfGACUAGACsAsCdTdT 3';

[0486] DDP0405-DS00702:

[0487] siRNA1: Sense = 5' GsCsUGAGfGAfGfAfAUUGCUUCAsUsA 3';

[0488] Antisense = 5' VPUsdCsUCdAAdTUAAGdTUfGACUAGACsAsCdTdT 3';

[0489] siRNA2: Sense = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0490] Antisense = 5' VPUsdCsUCdAAdTUAAGdTUfGACUAGACsAsCdTdT 3';

[0491] DDP05-DS00701:

[0492] siRNA1: Sense = 5' GsCsUGAGfGAfGfAfAUUGCUUCAsUsA 3';

[0493] Antisense = 5' VPUsdCsUCdAAdTUAAGdTUfGACUAGACsAsCdTdT 3';

[0494] siRNA2: sense strand = 5' GsUsCUAGfUCfAfAfCUUAAUUGAsGsA 3';

[0495] antisense strand = 5' UsdCsUCdAAdTUAAGdTUfGACUAGACsAsC 3';

[0496] DDP0501-DS00701:

[0497] siRNA1: sense strand = 5' GsCsUGAGfGAfGfAfAUUGCUUCAsUsA 3';

[0498] antisense strand = 5' VPUsfAsUGAfAGfCAAUUCfUCfCUCAGCsAsC 3';

[0499] siRNA2: sense strand = 5' GsUsCUAGfUCfAfAfCUUAAUUGAsGsA 3';

[0500] antisense strand = 5' VPUsdCsUCdAAdTUAAGdTUfGACUAGACsAsC 3';

[0501] DDP0502-DS00701:

[0502] siRNA1: sense strand = 5' GsCsUGAGfGAfGfAfAUUGCUUCAsUsA 3';

[0503] antisense strand = 5' UsfAsUGAfAGfCAAUUCfUCfCUCAGCsAsCdTdT 3';

[0504] siRNA2: sense strand = 5' GsUsCUAGfUCfAfAfCUUAAUUGAsGsA 3';

[0505] antisense strand = 5' UsdCsUCdAAdTUAAGdTUfGACUAGACsAsCdTdT 3';

[0506] DDP05-DS00703:

[0507] siRNA1: sense strand = 5' AsGsAAAUfUCfUfAfCUACAUCUAsUsA 3';

[0508] antisense strand = 5' UsdAsUAdGATGUAGTAfGAAUUUCUsCsU 3';

[0509] siRNA2: sense strand = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0510] antisense strand = 5' AsdAsAUTCdAGGAATUfCCUGCUUCsUsU 3';

[0511] DDP0502-DS00702:

[0512] siRNA1: sense strand = 5' GsCsUGAGfGAfGfAfAUUGCUUCAsUsA 3';

[0513] antisense strand = 5' UsfAsUGAfAGfCAAUUCfUCfCUCAGCsAsCdTdT 3';

[0514] siRNA2: sense strand = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0515] antisense strand = 5' AsdAsAUdTCdAGGAAdTUfCCUGCUUCsUsUdTdT 3';

[0516] DDP06-DS00101:

[0517] siRNA1: sense strand = 5' UsUsGUCCfCAfGfUfAUUCUAUGUsUsU 3';

[0518] antisense strand = 5' AsdAsACdAUdAGAAUdACfUGGGACAAsCsG 3';

[0519] siRNA2: sense strand = 5' GsCsUGAGfGAfGfAfAUUGCUUCAsUsA 3';

[0520] antisense strand = 5' UsfAsUGAfAGfCAAUUCfUCfCUCAGCsAsC 3';

[0521] DDP06-DS00703:

[0522] siRNA1: sense strand = 5' AsGsAAAUfUCfUfAfCUACAUCUAsUsA 3';

[0523] antisense strand = 5' UsdAsUAdGATGUAGTAfGAAUUUCUsCsU 3';

[0524] siRNA2: sense strand = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0525] antisense strand = 5' AsdAsAUTCdAGGAATUfCCUGCUUCsUsU 3';

[0526] DDP0602-DS00702:

[0527] siRNA1: sense strand = 5' GsCsUGAGfGAfGfAfAUUGCUUCAsUsA 3';

[0528] antisense strand = 5' UsfAsUGAfAGfCAAUUCfUCfCUCAGCsAsCdTdT 3';

[0529] siRNA2: sense strand = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0530] antisense strand = 5' AsdAsAUdTCdAGGAAdTUfCCUGCUUCsUsUdTdT 3';

[0531] DDP07-DS00703:

[0532] siRNA1: sense strand = 5' AsGsAAAUfUCfUfAfCUACAUCUAsUsA 3';

[0533] antisense strand = 5' UsdAsUAdGATGUAGTAfGAAUUUCUsCsU 3';

[0534] siRNA2: sense strand = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0535] antisense strand = 5' AsdAsAUTCdAGGAATUfCCUGCUUCsUsU 3';

[0536] DDP0702-DS00702:

[0537] siRNA1: sense strand = 5' GsCsUGAGfGAfGfAfAUUGCUUCAsUsA 3';

[0538] Sense = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0539] Sense = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0540] Sense = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0541] DDP08-DS00401:

[0542] Sense = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0543] Sense = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0544] Sense = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0545] Sense = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0546] DDP09-DS00401:

[0547] Sense = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0548] Sense = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0549] Sense = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0550] Sense = 5' GsAsAGCAfGGfAfAfUUCCUGAAUsUsU 3';

[0551] DDP10-DS00401:

[0552] siRNA1 : sense strand = 5' CsUsAGACfCUfGUdTUUGCUUUUsGsU 3';

[0553] antisense strand = 5' VPAsfCsAfAfAfAGfCAfAAfACfAGfGUfCUAGsAsAdTdT 3';

[0554] siRNA2: sense strand = 5' GsAsGCGACAfGfUfAUUCUCAGUsGsU 3';

[0555] antisense strand = 5' VPAsfCsAfCUfGAfGAAUACfUGfUCfGCsfUsCdTdT 3';

[0556] DDP11-DS00401:

[0557] siRNA1 : sense strand = 5' CsUsAGACfCUfGUdTUUGCUUUUsGsU 3';

[0558] antisense strand = 5' VPAsfCsAfAfAfAGfCAfAAfACfAGfGUfCUAGsAsAdTdT 3';

[0559] siRNA2: sense strand = 5' GsAsGCGACAfGfUfAUUCUCAGUsGsU 3';

[0560] antisense strand = 5' VPAsfCsAfCUfGAfGAAUACfUGfUCfGCsfUsCdTdT 3';

[0561] DDP08-DS00501:

[0562] siRNA1 : sense strand = 5' CsUsAGACfCUfGUdTUUGCUUUUsGsU 3';

[0563] antisense strand = 5' VPAsfCsAfAfAfAGfCAfAAfACfAGfGUfCUAGsAsAdTdT 3';

[0564] siRNA2: sense strand = 5' CsAsGCUCCUfUfAfUUGUUAUACsGsA 3';

[0565] antisense strand = 5' VPUsfCsGfUAfUAACAAfUAfAGfGAfGCsfUsGdTdT 3';

[0566] DDP09-DS00501:

[0567] siRNA1: sense strand = 5' CsUsAGACfCUfGUdTUUGCUUUUsGsU 3';

[0568] antisense strand = 5' VPAsfCsAfAfAfAGfCAfAAfACfAGfGUfCUAGsAsAdTdT 3';

[0569] siRNA2: sense strand = 5' CsAsGCUCCUfUfAfUUGUUAUACsGsA 3';

[0570] antisense strand = 5' VPUsfCsGfUAfUAACAAfUAfAGfGAfGCsfUsGdTdT 3';

[0571] DDP10-DS00501:

[0572] siRNA1: sense strand = 5' CsUsAGACfCUfGUdTUUGCUUUUsGsU 3';

[0573] antisense strand = 5' VPAsfCsAfAfAfAGfCAfAAfACfAGfGUfCUAGsAsAdTdT 3';

[0574] siRNA2: sense strand = 5' CsAsGCUCCUfUfAfUUGUUAUACsGsA 3';

[0575] antisense strand = 5' VPUsfCsGfUAfUAACAAfUAfAGfGAfGCsfUsGdTdT 3';

[0576] DDP11-DS00501:

[0577] siRNA1: sense strand = 5' CsUsAGACfCUfGUdTUUGCUUUUsGsU 3';

[0578] antisense strand = 5' VPAsfCsAfAfAfAGfCAfAAfACfAGfGUfCUAGsAsAdTdT 3';

[0579] siRNA2: sense strand = 5' CsAsGCUCCUfUfAfUUGUUAUACsGsA 3';

[0580] Sense strand = 5' VPUsfCsGfUAfUAACAAfUAfAGfGAfGCsfUsGdTdT 3';

[0581] DDP08-DS006:

[0582] siRNA1 : Sense strand = 5' CsUsAGACfCUfGUdTUUGCUUUUsGsU 3';

[0583] Antisense strand = 5' AsfCsAfAfAfAGfCAfAAfACfAGfGUfCUAGsAsAdTdT 3';

[0584] siRNA2: Sense strand = 5' CsUsCAACAUfAfUfUUGAUCAGUsCsU 3';

[0585] Antisense strand = 5' AsdGsACdTGdAUCAAdAUfAUGUUGAGsCsUdTdT 3';

[0586] DDP08-DS00611:

[0587] siRNA1 : Sense strand = 5' CsUsAGACfCUfGUdTUUGCUUUUsGsU 3';

[0588] Antisense strand = 5' VPAsfCsAfAfAfAGfCAfAAfACfAGfGUfCUAGsAsA 3';

[0589] siRNA2: Sense strand = 5' CsUsCAACAUfAfUfUUGAUCAGUsCsU 3';

[0590] Antisense strand = 5' VPAsdGsACdTGdAUCAAdAUfAUGUUGAGsCsU 3';

[0591] DDP08-DS00612:

[0592] siRNA1 : Sense strand = 5' CsUsAGACfCUfGUdTUUGCUUUUsGsU 3';

[0593] Antisense strand = 5' VPAsfCsAfAfAfAGfCAfAAfACfAGfGUfCUAGsAsAdTdT 3';

[0594] siRNA2: sense strand = 5' CsUsCAACAUfAfUfUUGAUCAGUsCsU 3';

[0595] antisense strand = 5' VPAsdGsACdTGdAUCAAdAUfAUGUUGAGsCsU dTdT 3';

[0596] DDP09-DS006:

[0597] siRNA1: sense strand = 5' CsUsAGACfCUfGUdTUUGCUUUUsGsU 3';

[0598] antisense strand = 5' AsfCsAfAfAfAGfCAfAAfACfAGfGUfCUAGsAsAdTdT 3';

[0599] siRNA2: sense strand = 5' CsUsCAACAUfAfUfUUGAUCAGUsCsU 3';

[0600] antisense strand = 5' AsdGsACdTGdAUCAAdAUfAUGUUGAGsCsU dTdT 3';

[0601] DP09-DS00611:

[0602] siRNA1: sense strand = 5' CsUsAGACfCUfGUdTUUGCUUUUsGsU 3';

[0603] antisense strand = 5' VPAsfCsAfAfAfAGfCAfAAfACfAGfGUfCUAGsAsA 3';

[0604] siRNA2: sense strand = 5' CsUsCAACAUfAfUfUUGAUCAGUsCsU 3';

[0605] antisense strand = 5' VPAsdGsACdTGdAUCAAdAUfAUGUUGAGsCsU 3';

[0606] DDP09-DS00612:

[0607] siRNA1: sense strand = 5' CsUsAGACfCUfGUdTUUGCUUUUsGsU 3';

[0608] Antisense = 5' VPAsfCsAfAfAfAGfCAfAAfACfAGfGUfCUAGsAsAdTdT 3';

[0609] siRNA2: Sense = 5' CsUsCAACAUfAfUfUUGAUCAGUsCsU 3';

[0610] Antisense = 5' VPAsdGsACdTGdAUCAAdAUfAUGUUGAGsCsUdTdT 3';

[0611] DDP10-DS006:

[0612] siRNA1: Sense = 5' CsUsAGACfCUfGUdTUUGCUUUUsGsU 3';

[0613] Antisense = 5' AsfCsAfAfAfAGfCAfAAfACfAGfGUfCUAGsAsAdTdT 3';

[0614] siRNA2: Sense = 5' CsUsCAACAUfAfUfUUGAUCAGUsCsU 3';

[0615] Antisense = 5' AsdGsACdTGdAUCAAdAUfAUGUUGAGsCsUdTdT 3';

[0616] DDP11-DS006:

[0617] siRNA1: Sense = 5' CsUsAGACfCUfGUdTUUGCUUUUsGsU 3';

[0618] Antisense = 5' AsfCsAfAfAfAGfCAfAAfACfAGfGUfCUAGsAsAdTdT 3';

[0619] siRNA2: Sense = 5' CsUsCAACAUfAfUfUUGAUCAGUsCsU 3';

[0620] Antisense = 5' AsdGsACdTGdAUCAAdAUfAUGUUGAGsCsUdTdT 3';

[0621] HJY-0203-DS001:

[0622] siRNA1: sense strand = 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3';

[0623] antisense strand = 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3';

[0624] siRNA2: sense strand = 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3';

[0625] antisense strand = 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3';

[0626] HJY-0203-DS002:

[0627] siRNA1: sense strand = 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3';

[0628] antisense strand = 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3';

[0629] siRNA2: sense strand = 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3';

[0630] antisense strand = 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3';

[0631] HJY-0203-DS003:

[0632] siRNA1: sense strand = 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3';

[0633] antisense strand = 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3';

[0634] siRNA2: sense strand = 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3';

[0635] antisense strand = 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3';

[0636] HJY-0203-DS004:

[0637] siRNA1: sense strand = 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3';

[0638] antisense strand = 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3';

[0639] siRNA2: sense strand = 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3';

[0640] antisense strand = 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3';

[0641] HJY-0203-DS005:

[0642] siRNA1: sense strand = 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3';

[0643] antisense strand = 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3';

[0644] siRNA2: sense strand = 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3';

[0645] antisense strand = 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3';

[0646] HJY-0203-DS006:

[0647] siRNA1: sense strand = 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3';

[0648] antisense strand = 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3';

[0649] siRNA2: sense strand = 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3';

[0650] antisense strand = 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3';

[0651] HJY-0203-DS007:

[0652] siRNA1: sense strand = 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3';

[0653] antisense strand = 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3';

[0654] siRNA2: sense strand = 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3';

[0655] antisense strand = 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3';

[0656] HJY-0203-DS008:

[0657] siRNA1: sense strand = 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3';

[0658] antisense strand = 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3';

[0659] siRNA2: sense strand = 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3';

[0660] antisense strand = 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3';

[0661] HJY-0203-DS009:

[0662] siRNA1: sense strand = 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3';

[0663] antisense strand = 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3';

[0664] siRNA2: sense strand = 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3';

[0665] antisense strand = 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3';

[0666] HJY-0203-DS010:

[0667] siRNA1: sense strand = 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3';

[0668] antisense strand = 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3';

[0669] siRNA2: sense strand = 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3';

[0670] antisense strand = 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3';

[0671] HJY-0203-DS011:

[0672] siRNA1: sense strand = 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3';

[0673] antisense strand = 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3';

[0674] siRNA2: sense strand = 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3';

[0675] antisense strand = 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3';

[0676] HJY-0203-DS012:

[0677] siRNA1: sense strand = 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3';

[0678] antisense strand = 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3';

[0679] siRNA2: sense strand = 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3';

[0680] antisense strand = 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3';

[0681] HJY-0203-DS013:

[0682] siRNA1: sense strand = 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3';

[0683] antisense strand = 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3';

[0684] siRNA2: sense strand = 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3';

[0685] antisense strand = 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3';

[0686] HJY-0203-DS014:

[0687] siRNA1: sense strand = 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3';

[0688] antisense strand = 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3';

[0689] siRNA2: sense strand = 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3';

[0690] antisense strand = 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3';

[0691] HJY-0203-DS015:

[0692] siRNA1: sense strand = 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3';

[0693] antisense strand = 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3';

[0694] siRNA2: sense strand = 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3';

[0695] antisense strand = 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3';

[0696] HJY-0203-DS016:

[0697] siRNA1 : sense strand = 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3';

[0698] antisense strand = 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3';

[0699] siRNA2: sense strand = 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3';

[0700] antisense strand = 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3';

[0701] HJY-0203-DS017:

[0702] siRNA1 : sense strand = 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3';

[0703] antisense strand = 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3';

[0704] siRNA2: sense strand = 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3';

[0705] antisense strand = 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3';

[0706] HJY-0203-DS018:

[0707] siRNA1 : sense strand = 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3';

[0708] antisense strand = 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3';

[0709] siRNA2: sense strand = 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3';

[0710] antisense strand = 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3';

[0711] HJY-0203-DS019:

[0712] siRNA1: sense strand = 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3';

[0713] antisense strand = 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3';

[0714] siRNA2: sense strand = 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3';

[0715] antisense strand = 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3';

[0716] HJY-0203-DS020:

[0717] siRNA1: sense strand = 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3';

[0718] antisense strand = 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3';

[0719] siRNA2: sense strand = 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3';

[0720] antisense strand = 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3';

[0721] HJY-0203-DS021:

[0722] siRNA1: sense strand = 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3';

[0723] antisense strand = 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3';

[0724] siRNA2: sense strand = 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3';

[0725] antisense strand = 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3';

[0726] HJY-0203-DS022:

[0727] siRNA1: sense strand = 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3';

[0728] antisense strand = 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3';

[0729] siRNA2: sense strand = 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3';

[0730] antisense strand = 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3';

[0731] HJY-0203-DS023:

[0732] siRNA1: sense strand = 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3';

[0733] antisense strand = 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3';

[0734] siRNA2: sense strand = 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3';

[0735] antisense strand = 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3';

[0736] HJY-0203-DS024:

[0737] siRNA1: sense strand = 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3';

[0738] antisense strand = 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3';

[0739] siRNA2: sense strand = 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3';

[0740] antisense strand = 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3';

[0741] HJY-0203-DS025:

[0742] siRNA1 : sense strand = 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3';

[0743] antisense strand = 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3';

[0744] siRNA2: sense strand = 5' UsGsCAA(Ahd)UAfGfUfCUACAAACCsAsA 3';

[0745] antisense strand = 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3';

[0746] HJY-0203-DS026:

[0747] siRNA1 : sense strand = 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3';

[0748] antisense strand = 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3';

[0749] siRNA2: sense strand = 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3';

[0750] antisense strand = 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3';

[0751] HJY-0203-DS027:

[0752] siRNA1 : sense strand = 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3';

[0753] antisense strand = 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3';

[0754] siRNA2: sense strand = 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3';

[0755] antisense strand = 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3';

[0756] HJY-02-DS00:

[0757] Sense: 5' GsGsCUA(Chd)GAdAAdAUCCAACCUsAsA 3'

[0758] Antisense: 5'-VPUsfUsAGGU(Tgn)GGAUdTUfUCdGUAGCCsGsU 3', Chd is 2'-O-hexadecyl-cytidine-3'-phosphate, VP: vinyl-phosphate, Tgn: thymine-diol nucleic acid (GNA) S-isomer.

[0759] HJY-03-DS00:

[0760] Sense: 5' UsGsCAA(Ahd)fUAfGfUfCUACAAACCsAsA 3'

[0761] Antisense: 5'-VPUsfUsGGdTU(Tgn)GUAGACfUAfUUUGCAsCsA 3', Ahd is 2'-O-hexadecyl-adenosine-3'-phosphate, Tgn: thymine-diol nucleic acid (GNA) S-isomer.

[0762] G = 2'-O-methyl guanosine acid, A = 2'-O-methyl adenosine acid, U = 2'-O-methyl uridine acid, C = 2'-O-methyl cytidine acid; Gs = 2'-O-methyl-3'-thio guanosine acid, As = 2'-O-methyl-3'-thio adenosine acid, Us = 2'-O-methyl-3'-thio uridine acid, Ts = 2'-O-methyl-3'-thio thymidine acid, Cs = 2'-O-methyl-3'-thio cytidine acid; fG = 2'-fluoro guanosine acid, fA = 2'-fluoro adenosine acid, fU = 2'-fluoro uridine acid, fC = 2'-fluoro cytidine acid, fT = 2'-fluoro thymidine acid; fGs = 2'-fluoro-3'-thio guanosine acid, fAs = 2'-fluoro-3'-thio adenosine acid, fUs = 2'-fluoro-3'-thio uridine acid, fCs = 2'-fluoro-3'-thio cytidine acid, T = deoxythymidine acid, dA = 2'-deoxy adenosine acid, dG = 2'-deoxy guanosine acid, dT = 2'-deoxy thymidine acid.

[0763] In some embodiments, the sense strand or the antisense strand of the RNA inhibitor described herein is a sequence having at least 15 contiguous nucleotides identical to the sense strand or the antisense strand in Table 1, or a sequence differing by one, two or three nucleotides.

[0764] The sense strand and the antisense strand of the single delivery siRNA inhibitor used in the cell and animal experiments of the embodiments of the present application are shown in Table 1. The single delivery siRNA inhibitor is connected with a delivery carrier, and the single delivery siRNA inhibitor is shown in Table 3.

[0765] Table 3 siRNA inhibitor sequences and structures

[0766] The description and examples of L96 in patent WO2009082607 and WO2009073809, NAG25, NAG37 in WO2018044350A1, invAb in WO2021026150, invdA in WO2018140920 are all incorporated herein.

[0767] In some embodiments, the distribution, targeting, or stability of the RNA inhibitor is altered by the introduction of a delivery ligand in the carrier that targets a tissue receptor. For example, a specific ligand can provide enhanced affinity for a selected target (e.g., a molecule, a cell or cell type, a compartment (e.g., a cellular or organ compartment, a body tissue, an organ, or a region)) compared to a species in the absence of the ligand.

[0768] The delivery ligand can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low density lipoprotein (LDL), or globulin); a carbohydrate (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine, or hyaluronic acid); or a lipid. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid.

[0769] The delivery ligand can also include a targeting group, such as a cell or tissue targeting agent that binds to a specified cell type, such as a kidney cell, e.g., a lectin, glycoprotein, lipid, or protein, e.g., an antibody. The targeting group can be a thyroid stimulating hormone, melanocyte stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyamino acid, multivalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, cholic acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic. In some embodiments, the ligand is multivalent galactose, e.g., N-acetyl-galactosamine.

[0770] The sense and antisense strands of the RNA inhibitors described herein can be conveniently and routinely made using the standard techniques for oligonucleotide synthesis. Any other method known in the art for such synthesis, such as liquid phase synthesis or fermentation, can additionally or alternatively be used.

[0771] In some embodiments, in addition to the standard nucleoside phosphoramidite monomers that are commercially available and routinely used, and the non-standard nucleoside phosphoramidite monomers, the sense strand and the antisense strand of the RNA inhibitor of the present application can be synthesized by an automated synthesizer using the phosphoramidite method derived from the carrier-nucleoside phosphoramidite monomers.

[0772] 2.2 Synthesis of RNA inhibitor

[0773] The sense strand and the antisense strand of the uncoupled carrier structure are synthesized by using the standard solid-phase phosphoramidite method, using a multi-channel solid-phase synthesizer, and then the corresponding RNA inhibitor is prepared by complementary annealing of the sense strand and the antisense strand.

[0774] The basic steps of the solid-phase phosphoramidite method include:

[0775] 1) Deprotection: removing the Solid Support hydroxyl protecting group (DMTr) in the starting monomer;

[0776] 2) Coupling: adding the first phosphoramidite monomer, and coupling in the 3' to 5' direction;

[0777] 3) Oxidation: oxidizing the resulting nucleoside phosphite to a more stable nucleoside phosphate (i.e., trivalent phosphorus to pentavalent phosphorus);

[0778] 4) Blocking: blocking the 5'-OH of the failed nucleotide sequence in the previous step so that it no longer participates in further reactions; repeat the step until the last phosphoramidite monomer is added; then cleave the ester bond between the Solid Support and the starting monomer with aqueous methylamine and ammonia, and remove the protecting group on each base and the phosphate; after purification by HPLC, filter sterilize, and lyophilize to obtain the corresponding sense strand or antisense strand.

[0779] Description of the synthesis process of the RNA inhibitor:

[0780] Resuspend the sense strand and the antisense strand lyophilized powder separately, mix in equal molar amounts, add an appropriate amount of water for injection, and add an appropriate amount of TRIS buffer solution. Gently shake for about 1-2 min to mix the solution evenly. Warm the water bath to 92-95°C. Heat the reaction solution in the water bath for 3-5 min, and gently shake to evenly heat the solution. Naturally cool to room temperature. A colorless or slightly yellow transparent liquid is obtained, which is sampled for testing and concentration measurement.

[0781] Part III: RNA inhibitors for multiple targets

[0782] The RNA inhibitor of the present application can target multiple targets, and is mainly composed of a nucleotide chain, a linker chemical structure connecting two nucleotide chains, and a delivery ligand for delivering nucleotides into cells.

[0783] 3.1 The chemical structure of the linker consists of a orthogonal reactive group and a linker, the following examples are illustrated according to the type of orthogonal reactive group:

[0784] Example 3.1-1-1, the orthogonal reactive group is DBCO and azido compound:

[0785] Take C1, add 0.1M sodium tetraborate solution, completely dissolved; another C1-NHS ester is dissolved in DMSO, the solution is transferred to the nucleic acid solution, ultrasonic reaction for 2h, the control is qualified, add sodium acetate, centrifugal, sedimentation, drying to get 0.15g light yellow powder.

[0786] Take C2, add 0.1M sodium tetraborate solution, completely dissolved; another N3-C5 NHS ester is dissolved in DMSO, the solution is transferred to the nucleic acid solution, ultrasonic reaction for 2h, the control is qualified, add sodium acetate, centrifugal, sedimentation, drying to get 0.15g light yellow powder.

[0787] Take C3 and C4, add ultrapure water to dissolve. Stirring, the control is qualified, preparation and purification.

[0788] Example 3.1-1-2, the orthogonal reactive group is DBCO and azido compound:

[0789] Take C1, add Na2B4O7 solution, completely dissolved; another Azido-Aca-NHS (azido-C5-succinimidyl ester) is dissolved in DMSO, the solution is transferred to the nucleic acid solution, placed in 35℃ shaking bed reaction for 1h, the control is qualified, add NaOAc, centrifugal, sedimentation, drying to get powder C2.

[0790] Take C3, add Na2B4O7 solution, completely dissolved; another Azido-Aca-NHS (azido-C5-succinimidyl ester) is dissolved in DMSO, the solution is transferred to the nucleic acid solution, placed in 35℃ shaking bed reaction for 1h, the control is qualified, add NaOAc, centrifugal, sedimentation, drying to get powder C4.

[0791] Take linker08 dissolved in DMF, and take C2 dissolved in 3 mL of pure water, drop into the linker08 solution under stirring, stirring for 0.5 h, qualified in the control, directly prepare purification, concentrate to obtain the product DDP08-DBCO. Take DDP08-DBCO and C4, dissolve in ultrapure water, stir, qualified in the control, directly prepare purification, concentrate to obtain the product DDP08-S. Mix the product DDP-08-S with 3'-asRNA1-5', and 3'-asRNA2-5' in equal molar ratio in water for injection, heat to 70-95℃, then cool to room temperature, make it form double-stranded structure through hydrogen bond, to obtain the final product DDP08-DS.

[0792] Example 3.1-1-3, orthogonal reaction groups are DBCO and azide compounds:

[0793] Take C1, add Na2B4O7 solution, completely dissolved; take Azido-Aca-NHS (azido-C5-succinimidyl ester) dissolved in DMSO, transfer the solution to the nucleic acid solution, place it in a 35℃ shaking bed for 1 h, qualified in the control, add NaOAc, centrifuge, sediment, dry to obtain powder.

[0794] Take C3, add Na2B4O7 solution, completely dissolved; take Azido-Aca-NHS (azido-C5-succinimidyl ester) dissolved in DMSO, transfer the solution to the nucleic acid solution, place it in a 35℃ shaking bed for 1 h, qualified in the control, add NaOAc, centrifuge, sediment, dry to obtain powder.

[0795] Take linker09 dissolved in DMF, and take C2 dissolved in pure water, drop into the linker09 solution under stirring, stir, qualified in the control, directly prepare purification, concentrate, UV quantification, to obtain DDP09-DBCO.

[0796] Take DDP09-DBCO and C4, add ultrapure water to dissolve. Stir, qualified in the control, directly prepare purification, concentrate, UV quantification, to obtain DDP09-S.

[0797] Mix DDP09-S with 3'-RNA1-5' and 3'-RNA2-5' in equal molar ratio in water for injection, heat to 70-95℃, then cool to room temperature, make it form double-stranded structure through hydrogen bond, to obtain the final product DDP09-DS.

[0798] Example 3.1-1-4, orthogonal reaction groups are DBCO and azide compounds:

[0799] Take C1, add Na2B4O7 solution, completely dissolved; another take Azido-Aca-NHS (azido-C5-succinimidyl ester) is dissolved in DMSO, the solution is transferred to the nucleic acid solution, placed at 35°C shaking table reaction 1h, control qualified, add NaOAc, centrifugal, sedimentation, drying to get powder.

[0800] Take C3, add Na2B4O7 solution, completely dissolved; another take Azido-Aca-NHS (azido-C5-succinimidyl ester) is dissolved in DMSO, the solution is transferred to the nucleic acid solution, placed at 35°C shaking table reaction 1h, control qualified, add NaOAc, centrifugal, sedimentation, drying to get powder.

[0801] Take linker10 dissolved in DMF, another take C2 dissolved in pure water, drop in the solution of linker10 under stirring, stirring, control qualified, direct preparation purification, concentration, UV quantitative, get DDP09-DBCO.

[0802] Take DDP10-DBCO and C4, add ultrapure water to dissolve. Stirring, control qualified, direct preparation purification, concentration, UV quantitative, get DDP10-S.

[0803] DDP10-S and 3'-RNA1-5' and 3'-RNA2-5' are mixed in equal molar ratio in water for injection, heated to 70-95℃, then cooled to room temperature, so that it forms a double-stranded structure by hydrogen bonding, to get the final product DDP10-DS.

[0804] Example 3.1-1-5, orthogonal reaction group is DBCO and azide compound:

[0805] Take C1, add Na2B4O7 solution, completely dissolved; another take Azido-Aca-NHS (azido-C5-succinimidyl ester) is dissolved in DMSO, the solution is transferred to the nucleic acid solution, placed at 35°C shaking table reaction 1h, control qualified, add NaOAc, centrifugal, sedimentation, drying to get powder.

[0806] Take C3, add Na2B4O7 solution, completely dissolved; another take Azido-Aca-NHS (azido-C5-succinimidyl ester) is dissolved in DMSO, the solution is transferred to the nucleic acid solution, placed at 35°C shaking table reaction 1h, control qualified, add NaOAc, centrifugal, sedimentation, drying to get powder.

[0807] Take linker11 dissolved in DMF, another take C2 dissolved in pure water, drop in the solution of linker11 under stirring, stirring, control qualified, direct preparation purification, concentration, UV quantitative, get DDP09-DBCO.

[0808] Take DDP11-DBCO and C4, add ultrapure water to dissolve. Stir, control qualified, directly prepare purification, concentrate, UV quantification, get DDP11-S.

[0809] Mix DDP11-S with 3'-RNA1-5' and 3'-RNA2-5' in equimolar ratio in water for injection, heat to 70-95℃, then cool at room temperature, make it form double-stranded structure by hydrogen bond, get the final product DDP11-DS.

[0810] DBCO and azide compounds can be other structures besides the above structure, such as:

[0811] 1, Combination of.

[0812] 2, Combination of.

[0813] 3, Combination of.

[0814] 4, Combination of.

[0815] Example 3.1-2, orthogonal reaction group is mercapto and olefin

[0816] Take C1, add 0.1M sodium tetraborate solution, completely dissolved; another take E-3-benzoyl acrylate pentafluorobenzene ester dissolved in DMSO, transfer the solution to the nucleic acid solution, ultrasonic reaction for 2h, control qualified, add sodium acetate, centrifugal, sedimentation, drying to get light yellow powder.

[0817] Take C2, add 0.1M sodium tetraborate solution, completely dissolved; another take SATA dissolved in DMSO, transfer the solution to the nucleic acid solution, ultrasonic reaction for 2h, control qualified, add methylamine aqueous solution, control qualified, add sodium acetate, centrifugal, sedimentation, drying to get light yellow powder.

[0818] Take C3 and C4, add ultrapure water to dissolve. Stir, react in napi buffer (pH 8.0), control qualified, prepare purification.

[0819] Example 3.1-3: orthogonal reaction group is mercapto and olefin

[0820] Example 3.1-4: orthogonal reaction group is BCN and azide compound

[0821] Take C1, add 0.1M sodium tetraborate solution, completely dissolved; another take endo-BCN-NHS carbonate (Angew. Chem. Int. Ed. 2021, 60, 25905-25913) is dissolved in DMSO, the solution is transferred to the nucleic acid solution, ultrasonic reaction for 2h, the control is qualified, add sodium acetate, centrifugal, sedimentation, drying to get light yellow powder.

[0822] Take C2, add 0.1M sodium tetraborate solution, completely dissolved; another take N3-C5 NHS ester is dissolved in DMSO, the solution is transferred to the nucleic acid solution, ultrasonic reaction for 2h, the control is qualified, add methylamine aqueous solution, the control is qualified, add sodium acetate, centrifugal, sedimentation, drying to get light yellow powder.

[0823] Take C3 and C4, add ultrapure water to dissolve. Stir, react in napi buffer (pH 8.0), the control is qualified, prepare purification.

[0824] Example 3.1-5: Orthogonal reaction groups are phosphine and azide compounds

[0825] Take C1, add 0.1M sodium tetraborate solution, completely dissolved; another take AmAzCoupler is dissolved in DMSO, the solution is transferred to the nucleic acid solution, ultrasonic reaction for 2h, the control is qualified, add sodium acetate, centrifugal, sedimentation, drying to get g light yellow powder.

[0826] Take C2, add 0.1M sodium tetraborate solution, completely dissolved; another take N3-C5-NHS ester is dissolved in DMSO, the solution is transferred to the nucleic acid solution, ultrasonic reaction for 2h, the control is qualified, add sodium acetate, centrifugal, sedimentation, drying to get light yellow powder.

[0827] Take C3 and C4, add ultrapure water to dissolve, stir in napi buffer (pH 8.0), react at room temperature overnight, the control is qualified, prepare purification.

[0828] Example 3.1-6: Orthogonal reaction groups are TCO and tetrazine

[0829] Take C1, add 0.1M sodium tetraborate solution, completely dissolved; another take AmAzCoupler is dissolved in DMSO, the solution is transferred to the nucleic acid solution, ultrasonic reaction for 2h, the control is qualified, add sodium acetate, centrifugal, sedimentation, drying to get light yellow powder.

[0830] C2 was weighed and added to 0.1M sodium tetraborate solution and completely dissolved; N3-C5-NHS ester was weighed and dissolved in DMSO, and the solution was transferred to the nucleic acid solution, ultrasonic reaction was performed for 2h, and the control was qualified. Sodium acetate was added, centrifuged, precipitated, and dried to obtain a light yellow powder.

[0831] C3 and C4 were weighed and dissolved in ultrapure water, stirred in napi buffer (pH 8.0), and reacted at room temperature overnight. The control was qualified, and the product was purified.

[0832] 3.2 The RNA inhibitor of the application can target multiple targets, mainly composed of a nucleotide chain, a linker chemical structure connecting two nucleotide chains, and a delivery ligand for delivering nucleotides into cells.

[0833] The linker chemical structure is composed of an orthogonal reaction group and a connecting chain linker. The structure of the connecting chain linker between the orthogonal reaction group and the nucleotide chain is not limited, and can be composed of acyl, alkyl, thio, disulfide, PEG, or can be composed of homobifunctional linkers such as disuccinimidyl, bis-lactam, etc. The following examples are illustrated according to the structure type of the linker:

[0834] Example 3.2-1, the linker is composed of thio, acyl, and alkyl functional groups:

[0835] 2-(tritylthio)ethylamine (1.84 g) and Fmoc-Lys(N3)-OH (2.05 g) were weighed and dissolved in dichloromethane (30 mL), TBTU (2.19 g) and DIPEA (2.74 mL) were added, and the reaction was carried out at room temperature for 2 hours. Saturated sodium bicarbonate (30 mL) was added to quench the reaction, dichloromethane (30 mL*2) was extracted, the organic phase was washed with saturated brine (50 mL), anhydrous sodium sulfate was dried, filtered, rotary evaporated, and column purified to obtain the product.

[0836] The coupling product (2.50 g) was dissolved in dichloromethane (20 mL), diethylamine (1.0 mL) was added, and the reaction was carried out at room temperature for 2h. The solvent and diethylamine were removed by rotary evaporation, and then the product was dissolved in dichloromethane (20 mL), octanedioic acid monomethyl ester (0.82 g), TBTU (1.75 g) and DIPEA (1.90 mL) were added, and the reaction was carried out at room temperature overnight. Saturated sodium bicarbonate (20 mL) was added to quench the reaction, dichloromethane (20 mL*3) was extracted, the organic phase was combined, washed with saturated brine (30 mL), dried with anhydrous sodium sulfate, filtered, rotary evaporated, and column purified to obtain the product.

[0837] The coupling product (1.70 g) was dissolved in methanol (20 mL), water (10 mL) and lithium hydroxide (0.13 g) was added. After stirring at room temperature for 3 h, the methanol was removed by rotary evaporation. The residue was added with water (10 mL) and the pH was adjusted to 4.0 with 1 M HC1. The mixture was extracted with dichloromethane (20 mL x 3) and the organic phase was combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and rotary evaporated. The product was purified by column chromatography.

[0838] The carboxylic acid (1.50 g) and dlsanc-c12 (4.66 g) were dissolved in dichloromethane (50 mL), and TBTU (1.01 g) and DIPEA (1.25 mL) were added. The mixture was stirred at room temperature overnight. The reaction was quenched with saturated sodium bicarbonate (30 mL), and the aqueous phase was extracted with dichloromethane (50 mL x 2). The organic phase was combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and rotary evaporated. The product was purified by column chromatography.

[0839] The coupling product (2.50 g) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (10 mL) was added. Triethylsilane (0.50 mL) was added dropwise, and the mixture was stirred at room temperature for 1 h. The solvent, trifluoroacetic acid and triethylsilane were removed by rotary evaporation. The residue was dissolved in dichloromethane (10 mL) and rotary evaporated three times. The crude product was obtained by oil pump drying.

[0840] DDP01-DS06-C1 was weighed and dissolved in 0.1 M sodium tetraborate solution. N-hydroxysuccinimidyl iodoacetate dissolved in DMSO was added to the nucleic acid solution, and the mixture was ultrasonically reacted for 2 h. After passing the control, sodium acetate was added, centrifuged, settled and dried to obtain a light yellow powder.

[0841] DDP01-DS06-C1 was weighed and dissolved in 0.1 M sodium tetraborate solution. DBCO-NHS ester dissolved in DMSO was added to the nucleic acid solution, and the mixture was ultrasonically reacted for 2 h. After passing the control, sodium acetate was added, centrifuged, settled and dried to obtain a light yellow powder.

[0842] DDP01-DS06-C6 and DDP01-DS06-C2 were weighed and dissolved in ultrapure water. After stirring, napi buffer (pH 8.0) was added and the mixture was stirred at room temperature overnight. After passing the control, sodium hydroxide aqueous solution was added to remove the acetyl group. After complete removal, the product was purified by preparation.

[0843] Example 3.2-2, the linker is composed of disulfide bond, acyl group and alkyl group.

[0844] Dithio-pyridine (1.47 g) and Fmoc-Lys(N3)-OH (2.46 g) were weighed and dissolved in dichloromethane (30 mL), TBTU (2.63 g) and DIPEA (3.29 mL) were added, and the reaction was allowed to proceed at room temperature for 2 hours. Saturated sodium bicarbonate (30 mL) was added to quench the reaction, and dichloromethane (30 mL*2) was used to extract the organic phase. The organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and rotary evaporated. The product was purified by column chromatography.

[0845] The coupling product (2.75 g) was dissolved in dichloromethane (20 mL), diethylamine (1.0 mL) was added, and the reaction was allowed to proceed at room temperature for 2 hours. The solvent and diethylamine were removed by rotary evaporation, and the residue was dissolved in dichloromethane (20 mL). Octanedioic acid monomethyl ester (1.05 g), TBTU (1.93 g), and DIPEA (2.10 mL) were added, and the reaction was allowed to proceed at room temperature overnight. Saturated sodium bicarbonate (20 mL) was added to quench the reaction, and dichloromethane (20 mL*3) was used to extract the organic phase. The combined organic phase was washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and rotary evaporated. The product was purified by column chromatography.

[0846] The coupling product (1.70 g) was dissolved in methanol (20 mL), water (10 mL) was added, and lithium hydroxide (0.13 g) was added. The reaction was allowed to proceed at room temperature for 3 hours, and the methanol was removed by rotary evaporation. Water (10 mL) was added to the residue, and the pH was adjusted to 4.0 with 1M HCl. The reaction was extracted with dichloromethane (20 mL*3), and the combined organic phase was washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and rotary evaporated. The product was purified by column chromatography.

[0847] Carboxylic acid (1.59 g) and dlsanc-c12 (5.92 g) were weighed and dissolved in dichloromethane (50 mL). TBTU (1.07 g) and DIPEA (1.33 mL) were added, and the reaction was allowed to proceed at room temperature overnight. Saturated sodium bicarbonate (30 mL) was added to quench the reaction, and the aqueous phase was extracted with dichloromethane (50 mL*2). The combined organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and rotary evaporated. The product was purified by column chromatography.

[0848] Example 3.2-3: Linker containing bis-succinimidyl octanedioate

[0849] Fmoc-Lys(N3)-OH (1.13 g) and tri-fork-amino hexanoic acid (CN111116684A disclosed) (4.51 g) were dissolved in dichloromethane (50 mL), TBTU (1.09 g) and DIPEA (1.36 mL) were added, and the reaction was allowed to proceed at room temperature overnight. Saturated sodium bicarbonate (50 mL) was added to quench the reaction, and the aqueous phase was extracted with dichloromethane (50 mL*2). The combined organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and rotary evaporated. The product was purified by column chromatography.

[0850] The coupling product (2.75 g) was dissolved in dichloromethane (20 mL), diethylamine (1.0 mL) was added, and the reaction was allowed to proceed at room temperature for 2 h. The solvent and diethylamine were removed by rotary evaporation, and the residue was dried by oil pump and dissolved in DMSO.

[0851] DDP01-DS06-C1 was weighed and dissolved in 0.1 M sodium tetraborate solution. Another solution of bis-succinimidyl glutarate in DMSO was prepared and transferred into the nucleic acid solution. The reaction was allowed to proceed for 1 h. After the reaction was completed, sodium acetate was added, and the mixture was centrifuged, precipitated, and dried to obtain a light yellow powder.

[0852] DDP01-DS06-C1-activated ester was weighed and dissolved in 0.1 M sodium tetraborate solution. Another solution of tris-azido acid in DMSO was prepared and transferred into the nucleic acid solution. The reaction was allowed to proceed for 1 h. After the reaction was completed, sodium acetate was added, and the mixture was centrifuged, precipitated, and dried to obtain a light yellow powder.

[0853] DDP01-DS06-C6 and DDP01-DS06-C2 were weighed and dissolved in ultrapure water. The solution was stirred in napi buffer (pH 8.0) at room temperature overnight. After the reaction was completed, sodium hydroxide solution was added to remove the acetyl group. After complete removal, the product was purified by preparative purification.

[0854] Example 3.2-4: Linker containing PEG, bis-lactam:

[0855] 3.3 The RNA inhibitor of the present application can target multiple targets, mainly composed of nucleotide chains, linker chemical structure connecting two nucleotide chains, and delivery ligand for delivering nucleotides into cells.

[0856] The delivery ligand can be attached to the interior or the end of the nucleotide, or can be attached to the linker chemical structure through a linking chain. As one embodiment, the 5' end of the sense strand of the double-stranded small interfering nucleic acid is linked in series through the linker chemical structure, and the 3' end or interior of the sense strand is conjugated with the delivery ligand structure or the 3' end or interior of the antisense strand paired with the sense strand is conjugated with the delivery ligand. As another embodiment, the 3' end of the sense strand of the double-stranded small interfering nucleic acid is linked in series through the linker chemical structure, and the 5' end or interior of the sense strand is conjugated with the ligand or lipophilic structure or the 5' end or interior of the antisense strand paired with the sense strand is conjugated with the delivery ligand. As another embodiment, the 3' end of the antisense strand of the double-stranded small interfering nucleic acid is linked in series through the linker chemical structure, and the 5' end or interior of the sense strand paired with the antisense strand is conjugated with the delivery ligand. Wherein, the conjugation with the interior of the sense strand or the antisense strand refers to the conjugation with the nucleotide in the interior, and as one embodiment, the conjugation site can be the 3' position of the ribose five-membered ring of one of the nucleotides in the nucleotide chain, and can be conjugated with one or multiple, without limitation, and the position of the nucleotide in the nucleotide chain is also not limited, which can be the 6th position from the 5' end, or other positions.

[0857] The multi-target inhibitor aims to solve the problem that one drug can be used to treat multiple gene diseases, such as cardiovascular diseases or neurological diseases, and the present application can bring multiple nucleic acid drugs into the same cell through one compound. Compared with the inhibitor composition, the present application can ensure that two or more siRNAs are simultaneously taken up by the same cell, and can potentially simplify the development path of the multi-inhibitor mixture, and can avoid multiple administrations and reduce the pain of patients. The linker of the present application is expected to improve the delivery or pharmacokinetic characteristics of the multi-target inhibitor. The linker selected in the present application has excellent biocompatibility and high safety.

[0858] The type of delivery ligand is not limited, as long as it can deliver the drug into the cell, and is suitable for the present application.

[0859] 3.4 Multi-target RNA inhibitor synthesis process:

[0860] 3.4.1 Example 3.4-1 DDP04-DS001 synthesis:

[0861] 3.4.1.1 Synthesis of Kylo-19-S003-MMT

[0862] wherein the sequence of the sense strand of siRNA1 is 5' UsUsGUCCfCAfGfUfAUUCUAUGUsUsU 3'

[0863] The nucleotide monomers were added one at a time in the order of the sequence described above by the phosphoramidite solid phase synthesis method. Each addition of a nucleotide monomer included a deprotection, coupling, capping, and oxidation reaction, and the cycle was repeated 22 times. The synthesis conditions are described below:

[0864] The nucleotide monomers were dissolved in acetonitrile, and the conditions for the deprotection reaction were the same for each cycle, i.e., a temperature of 25°C, a reaction time of 45 seconds, a deprotection reagent of dichloroacetic acid in dichloromethane (3% v / v), and a molar ratio of dichloroacetic acid to the 4,4' dimethoxytrityl protecting group on the solid support of 4:1. The conditions for the coupling reaction were the same for each cycle, i.e., a temperature of 25°C, a molar ratio of the sequence on the solid support to the nucleotide monomer of 1:4, a molar ratio of the sequence on the solid support to the coupling reagent of 1:65, a reaction time of 300 seconds, and a coupling reagent of 5-ethylthio-1H-tetrazole in 0.25 M acetonitrile. The conditions for the capping were the same for each cycle, i.e., a temperature of 25°C and a reaction time of 15 seconds. The capping reagent solution was a mixture of Cap 1 and Cap 2 in a molar ratio of 1:1, and the molar ratio of the capping reagent to the sequence on the solid support was acetic anhydride:N-methylimidazole:sequence on the solid support = 1:1:1. The conditions for the oxidation reaction were the same for each cycle, i.e., a temperature of 25°C, a reaction time of 15 seconds, and an oxidation reagent of 0.05 M iodine in water. The molar ratio of iodine to the sequence on the solid support from the coupling step was 25:1. The reactions were carried out in a mixture of tetrahydrofuran:water:pyridine = 3:1:1. When the target sequence included a phosphorothioate linkage between two nucleotides, the oxidation reaction step was replaced with the following thiolation reaction step for the addition of the last of the two nucleotides; the conditions for each thiolation reaction were the same, i.e., a temperature of 25°C, a reaction time of 300 seconds, and a thiolation reagent of hydriodixone. The molar ratio of the thiolation reagent to the sequence on the solid support from the coupling step was 100:1. The reactions were carried out in a mixture of acetonitrile:pyridine = 1:1.

[0865] 3.4.1.2. Synthesis of Kylo-27-S004-MMT:

[0866] wherein the sequence of the sense strand of siRNA 2 is 5' AsGsAAAUfUCfUfAfCUACAUCUAsUsA 3'

[0867] The nucleotide monomers were added one at a time in the order of the sequence described above by the phosphoramidite solid phase synthesis method. The conditions for the deprotection, coupling, capping, and oxidation reactions were the same as described in 1.1, and the cycle was repeated 22 times.

[0868] 3.4.1.3. Synthesis of Kylo-19-AS003-3' MVIP09

[0869] Antisense strand sequence of siRNA1: 5' AsdAsACdAUdAGAAUdACfUGGGACAAsCsG 3'

[0870] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 1.1 were adopted, including the conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method. The reaction was recycled for 23 times.

[0871] 3.4.1.4 Synthesis of Kylo-27-AS004-3'MVIP09

[0872] Antisense strand sequence of siRNA2: 5' UsdAsUAdGATGUAGTAfGAAUUUCUsCsU 3'

[0873] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 1.1 were adopted, including the conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method. The reaction was recycled for 23 times.

[0874] 3.4.1.5 Synthesis of DDP04-S001-C2:

[0875] Kylo-19-S003-MMT (0.3 g, 0.04 mmol) was weighed and dissolved in 2 mL of ultrapure water, 2 volumes of ice HOAc were added and mixed, then placed in a 35°C shaking bed for 1 h of reaction, and the control was qualified, Na2CO3 was added, settled, dried to obtain 0.20 g of white powder, yield 70.19%. MS m / z: C218H286F4N65O148P21S4, [M+1]+, theoretical: 7039.67, found: 7039.06.

[0876] DDP04-S001-C1 (0.20 g, 0.028 mmol) was weighed and added with 0.1 M Na2B4O7 solution (1 mL), completely dissolved; Azido-Aca-NHS (0.022 g, 0.09 mmol, azido-C5-succinimidyl ester) was dissolved in 1 mL of DMSO, and the solution was transferred to the nucleic acid solution, placed in a 35°C shaking bed for 1 h of reaction, and the control was qualified, NaOAc was added, centrifuged, settled, dried to obtain 0.14 g of light yellow powder, yield 68.64%. MS m / z: C224H295F4N68O149P21S4, [M+1]+, theoretical: 7178.83, found: 7178.13.

[0877] 3.4.1.6 Synthesis of DDP04-DS001-C4:

[0878] Take Kylo-27-S004-MMT (0.3g, 0.04mmol) dissolved in 2mL ultrapure water, add 2 volumes of ice HOAc, mix well, and then place it in a 35°C shaking bed for 1h. After the control is qualified, add Na2CO3, settle, and dry to obtain 0.20g white powder, with a yield of 73.67%. MS m / z: C222H290F4N77O138P21S4, [M+1]+, theoretical: 7099.84, found: 7099.12.

[0879] Take DDP04-S001-C1 (0.20g, 0.03mmol) and add 0.1M Na2B4O7 solution (1mL) to completely dissolve it. Then take DBCO-NHS ester (0.036g, 0.09mmol, azadibenzocyclooctyne-active ester) dissolved in 1mL DMSO, and transfer the solution to the nucleic acid solution. Place it in a 35°C shaking bed for 1h. After the control is qualified, add NaOAc, centrifuge, settle, and dry to obtain 0.14g light yellow powder, with a yield of 64.07%. MS m / z: C241H303F4N78O140P21S4, [M+1]+, theoretical: 7387.16, found: 7386.85. The mass spectrum is shown in Figure 1.

[0880] 3.4.1.7 Synthesis of DDP04-S001

[0881] Take DDP04-S001-C2 (0.05g, 0.007mmol) and DDP41-S01-C4 (0.061g, 0.008mmol), and add 3mL ultrapure water to dissolve them. Stir for 4h, and after the control is qualified, directly prepare and purify them. Concentrate to 1mL, and quantitatively determine them by UV to obtain 38.1mg of DDP04-S001, with a yield of 37.46%. MS m / z: C465H597F8N146O289P42S8, [M+1]+, theoretical: 14565.99, found: 14565.24.

[0882] 3.4.1.8 Synthesis of DDP04-DS001

[0883] Mix DDP04-S001 with Kylo-19-AS003 and Kylo-27-AS004 in an equimolar ratio in water for injection, heat to 70-95°C, then cool to room temperature, and allow it to form a double-stranded structure through hydrogen bonding to obtain the final product DDP04-DS001. DDP04-DS002-DDP04-DS006 are also synthesized according to the above method.

[0884] The key steps in the synthesis of the DDP05, DDP06 and DDP07 series of compounds are similar to those of the DPP04 compound above.

[0885] 3.4.2 Example 3.4-2 Synthesis of HJY-0203-DS008

[0886] 3.4.2.1 Synthesis of HJY-02-S001-MMT

[0887] wherein the sequence of the sense strand of siRNA1 is 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3'

[0888] The nucleotide monomers were added in the order of the sequence from 3' to 5' by the phosphoramidite solid phase synthesis method. Each addition of a nucleotide monomer included a deprotection, a coupling, a capping, and an oxidation reaction. The cycle was repeated 22 times. The synthesis conditions are described below.

[0889] The nucleotide monomers were dissolved in acetonitrile. The conditions for the deprotection reaction of each cycle were the same, i.e., a temperature of 25 °C, a reaction time of 45 seconds, a deprotection reagent of dichloroacetic acid in dichloromethane (3% v / v), and a molar ratio of dichloroacetic acid to the 4,4' dimethoxytrityl protecting group on the solid support of 4:1. The conditions for the coupling reaction of each cycle were the same, i.e., a temperature of 25 °C, a molar ratio of the sequence on the solid support to the nucleotide monomer of 1:4, a molar ratio of the sequence on the solid support to the coupling reagent of 1:65, a reaction time of 300 seconds, and a coupling reagent of 5-ethylthio-1H-tetrazole in 0.25 M acetonitrile. The conditions for the capping of each cycle were the same, i.e., a temperature of 25 °C and a reaction time of 15 seconds. The capping reagent solution was a mixture of Cap1 and Cap2 at a molar ratio of 1:1. The molar ratio of the capping reagent to the sequence on the solid support was acetic anhydride:N-methylimidazole:sequence on the solid support = 1:1:1. The conditions for the oxidation reaction of each cycle were the same, i.e., a temperature of 25 °C, a reaction time of 15 seconds, and an oxidation reagent of 0.05 M iodine in water. The molar ratio of iodine to the sequence on the solid support from the coupling step was 25:1. The reaction was carried out in a mixture of tetrahydrofuran:water:pyridine = 3:1:1. When the target sequence had a phosphorothioate linkage between two nucleotides, the oxidation reaction step in the coupling of the latter nucleotide was replaced by the following thiolation reaction step. The conditions for each thiolation reaction step were the same, i.e., a temperature of 25 °C, a reaction time of 300 seconds, and a thiolation reagent of hydriodic acid. The molar ratio of the thiolation reagent to the sequence on the solid support from the coupling step was 100:1. The reaction was carried out in a mixture of acetonitrile:pyridine = 1:1.

[0890] 3.4.2.2 Synthesis of HJY-03-S001-MMT:

[0891] The sequence of the sense strand of siRNA2 is 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3'

[0892] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method. The reaction was cycled for 22 times.

[0893] 3.4.2.3 Synthesis of HJY-02-AS001-dp01

[0894] The sequence of the antisense strand of siRNA1 is 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3'

[0895] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method. The reaction was cycled for 23 times.

[0896] 3.4.2.4 Synthesis of HJY-03-AS001-dp02

[0897] The sequence of the antisense strand of siRNA2 is 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3'

[0898] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method. The reaction was cycled for 23 times.

[0899] 3.4.2.5 Synthesis of HJY-0203-C2:

[0900] HJY-02-S001-MMT (0.4 g) was dissolved in 2 mL of ultrapure water, mixed with 2 volumes of ice HOAc, and placed on a shaking table at 35°C for 1 h. After passing the control, Na2CO3 was added, settled, and dried to obtain 0.31 g of white powder with a yield of 81.51%. MS m / z: C225H297F3N85O135P21S4, [M+1]+, theoretical: 7178.99, actual: 7178.26.

[0901] Take HJY-0203-C1 (0.31 g), add 0.1 M Na2B4O7 solution (1 mL), completely dissolved; another take Azido-Aca-NHS (0.033 g, azido-C5-succinimidyl ester) dissolved in 1 mL DMSO, transfer the solution to the nucleic acid solution, place it in a 35°C shaking bed for 1 h, control qualified, add NaOAc, centrifuge, sediment, dry to obtain 0.25 g of light yellow powder, yield 79.11%. MS m / z: C231H306F3N88O136P21S4, [M+1]+, theoretical: 7327.15, found: 7326.84.

[0902] 3.4.2.6 Synthesis of HJY-0203-C4:

[0903] Take HJY-03-S001-MMT (0.4 g) and add 2 mL of ultrapure water to dissolve, mix well after adding 2 volumes of ice HOAc, place it in a 35°C shaking bed for 1 h, control qualified, add Na2CO3, sediment, dry to obtain 0.33 g of white powder, yield 86.78%. MS m / z: C225H296F3N84O135P21S4, [M+1]+, theoretical: 7172.98, found: 7172.32.

[0904] Take HJY-0203-C3 (0.33 g), add 0.1 M Na2B4O7 solution (1 mL), completely dissolved; another take DBCO-NHS ester (0.055 g, diazabicyclooctyne-active ester) dissolved in 1 mL DMSO, transfer the solution to the nucleic acid solution, place it in a 35°C shaking bed for 1 h, control qualified, add NaOAc, centrifuge, sediment, dry to obtain 0.26 g of light yellow powder, yield 75.75%. MS m / z: C244H309F3N85O137P21S4, [M+1]+, theoretical: 7460.30, found: 7760.01.

[0905] 3.4.2.7 Synthesis of HJY-0203-SS:

[0906] Take HJY-0203-C2 (0.2 g) and HJY-0203-C4 (0.244 g), add 2 mL of ultrapure water to dissolve. Stir for 4 h, control qualified, directly prepare and purify, concentrate to 1 mL, UV quantification, obtain 151 mg of HJY-0203-SS, yield 37.16%. MS m / z: C465H597F8N146O289P42S8, [M+1]+, theoretical: 14787.45, found: 14787.06.

[0907] 3.4.2.8 Synthesis of HJY-0203-DS008

[0908] HJY-0203-SS was mixed with HJY-02-AS001-dp01 and HJY-03-AS001-dp02 in equimolar ratio in water for injection and heated to 70-95°C, then cooled to room temperature, allowing the formation of double-stranded structure by hydrogen bond, to obtain the final product HJY-0203-DS008.

[0909] 3.4.3 Example 3.4-3 Synthesis of HJY-0203-DS009

[0910] 3.4.3.1 Synthesis of HJY-02-S001-MMT

[0911] wherein the sequence of the sense strand of siRNA1 is 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3'

[0912] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method, and the reaction was cycled for 22 times.

[0913] 3.4.3.2 Synthesis of HJY-03-S001-MMT

[0914] wherein the sequence of the sense strand of siRNA2 is 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3'

[0915] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method, and the reaction was cycled for 22 times.

[0916] 3.4.3.3 HJY-02-AS001-dp02

[0917] The sequence of the antisense strand of siRNA1 is 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3'

[0918] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method. The reaction was cycled for 23 times.

[0919] 3.4.3.4 Synthesis of HJY-03-AS001-dp01

[0920] The sequence of the antisense strand of siRNA2 is: 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3'

[0921] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method. The reaction was cycled for 23 times.

[0922] 3.4.3.5 Synthesis of HJY-0203-C2

[0923] HJY-02-S001-MMT (0.4 g) was dissolved in 2 mL of ultrapure water, mixed with 2 volumes of ice HOAc, and placed on a shaking table at 35°C for 1 h. After passing the control, Na2CO3 was added, settled, and dried to obtain 0.31 g of white powder with a yield of 81.51%. MS m / z: C225H297F3N85O135P21S4, [M+1]+, theoretical: 7178.99, actual: 7178.26.

[0924] HJY-0203-C1 (0.31 g) was weighed and dissolved in 0.1 M Na2B4O7 solution (1 mL). Azido-Aca-NHS (0.033 g, 0.129 mmol, azido-C5-succinimidyl ester) was dissolved in 1 mL of DMSO, and the solution was transferred to the nucleic acid solution. It was placed on a shaking table at 35°C for 1 h. After passing the control, NaOAc was added, centrifuged, settled, and dried to obtain 0.25 g of light yellow powder with a yield of 79.11%. MS m / z: C231H306F3N88O136P21S4, [M+1]+, theoretical: 7327.15, actual: 7326.84.

[0925] 3.4.3.6 Synthesis of HJY-0203-C4

[0926] HJY-03-S001-MMT (0.4 g) was weighed into 2 mL of ultrapure water, mixed with 2 volumes of ice HOAc, and placed in a 35 °C shaker for 1 h. The control was qualified, Na2CO3 was added, and the mixture was settled and dried to obtain 0.33 g of white powder, with a yield of 86.78%. MS m / z: C225H296F3N84O135P21S4, [M+1]+, theoretical: 7172.98, actual: 7172.32.

[0927] HJY-0203-C3 (0.33 g) was weighed into 0.1 M Na2B4O7 solution (1 mL) and completely dissolved. DBCO-NHS ester (0.055 g azadibenzocyclooctyne-active ester) was dissolved in 1 mL DMSO, and the solution was transferred to the nucleic acid solution. The mixture was placed in a 35 °C shaker for 1 h. The control was qualified, NaOAc was added, and the mixture was centrifuged, settled, and dried to obtain 0.26 g of light yellow powder, with a yield of 75.75%. MS m / z: C244H309F3N85O137P21S4, [M+1]+, theoretical: 7460.30, actual: 7760.01.

[0928] 3.4.3.7 Synthesis of HJY-0203-SS

[0929] HJY-0203-C2 (0.2 g, 0.027 mmol) and HJY-0203-C4 (0.244 g, 0.033 mmol) were weighed into 2 mL of ultrapure water and dissolved by stirring for 4 h. The control was qualified, and the mixture was directly prepared and purified. After concentration to 1 mL, UV quantification was performed to obtain 151 mg of HJY-0203-SS, with a yield of 37.16%. MS m / z: C465H597F8N146O289P42S8, [M+1]+, theoretical: 14787.45, actual: 14787.06.

[0930] 3.4.3.8 Synthesis of HJY-0203-DS009

[0931] HJY-0203-SS, HJY-02-AS001-dp02, and HJY-03-AS001-dp01 were mixed in an equimolar ratio in water for injection, heated to 70-95 °C, and then cooled to room temperature. A double-stranded structure was formed by hydrogen bonding to obtain the final product HJY-0203-DS009.

[0932] 3.4.4 Synthesis of HJY-0203-DS010

[0933] 3.4.4.1 Synthesis of HJY-02-S001-MMT

[0934] Wherein, the sequence of the sense strand of siRNA1 is 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3'

[0935] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid phase synthesis. The same synthesis conditions as described in 3.4.1.1 were adopted, including the conditions of deprotection, coupling, capping, and oxidation in the solid phase synthesis method, and the reaction was cycled for 22 times.

[0936] 3.4.4.2 Synthesis of HJY-03-S001-MMT

[0937] Wherein, the sequence of the sense strand of siRNA2 is 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3'

[0938] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid phase synthesis. The same synthesis conditions as described in 3.4.1.1 were adopted, including the conditions of deprotection, coupling, capping, and oxidation in the solid phase synthesis method, and the reaction was cycled for 22 times.

[0939] 3.4.4.3 HJY-02-AS001-dp02

[0940] The sequence of the antisense strand of siRNA1 is 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3'

[0941] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid phase synthesis. The same synthesis conditions as described in 3.4.1.1 were adopted, including the conditions of deprotection, coupling, capping, and oxidation in the solid phase synthesis method. The reaction was cycled for 23 times.

[0942] 3.4.4.4 HJY-03-AS001

[0943] The sequence of the antisense strand of siRNA2 is 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3'

[0944] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid phase synthesis. The same synthesis conditions as described in 3.4.1.1 were adopted, including the conditions of deprotection, coupling, capping, and oxidation in the solid phase synthesis method. The reaction was cycled for 23 times.

[0945] 3.4.4.5 Synthesis of HJY-0203-C2:

[0946] HJY-02-S001-MMT (0.4 g, 0.053 mmol) was weighed into 2 mL of ultrapure water, dissolved, 2 volumes of ice HOAc were added, mixed, and then placed in a 35 °C shaker for 1 h. The control was qualified, Na2CO3 was added, settled, and dried to obtain 0.31 g of white powder, with a yield of 81.51%. MS m / z: C225H297F3N85O135P21S4, [M+1]+, theoretical: 7178.99, found: 7178.26.

[0947] HJY-0203-C1 (0.31 g, 0.043 mmol) was weighed, 0.1 M Na2B4O7 solution (1 mL) was added, and completely dissolved. Azido-Aca-NHS (0.033 g, 0.129 mmol, azido-C5-succinimidyl ester) was dissolved in 1 mL of DMSO, and the solution was transferred to the nucleic acid solution, placed in a 35 °C shaker for 1 h, and the control was qualified. NaOAc was added, centrifuged, settled, and dried to obtain 0.25 g of light yellow powder, with a yield of 79.11%. MS m / z: C231H306F3N88O136P21S4, [M+1]+, theoretical: 7327.15, found: 7326.84. The mass spectrum is shown in Figure 2.

[0948] 3.4.4.6 Synthesis of HJY-0203-C4:

[0949] HJY-03-S001-MMT (0.4 g, 0.053 mmol) was weighed into 2 mL of ultrapure water, dissolved, 2 volumes of ice HOAc were added, mixed, and then placed in a 35 °C shaker for 1 h. The control was qualified, Na2CO3 was added, settled, and dried to obtain 0.33 g of white powder, with a yield of 86.78%. MS m / z: C225H296F3N84O135P21S4, [M+1]+, theoretical: 7172.98, found: 7172.32.

[0950] Take HJY-0203-C3 (0.33g, 0.046mmol), add 0.1M Na2B4O7 solution (1mL), completely dissolved; another DBCO-NHS ester (0.055g, 0.138mmol, azadibenzocyclooctyne-active ester) was dissolved in 1mL DMSO, the solution was transferred to the nucleic acid solution, placed in a 35℃ shaking bed for 1h, the control was qualified, NaOAc was added, centrifuged, settled, dried to obtain 0.26g light yellow powder, yield 75.75%. MS m / z: C244H309F3N85O137P21S4, [M+1]+, the theoretical value: 7460.30, the measured value: 7760.01.

[0951] 3.4.4.7 Synthesis of HJY-0203-SS

[0952] Take HJY-0203-C2 (0.2g, 0.027mmol) and HJY-0203-C4 (0.244g, 0.033mmol), add 2mL ultrapure water to dissolve. Stir for 4h, the control is qualified, directly prepare purification, concentrate to 1mL, UV quantification, obtain 151mg of HJY-0203-SS, yield 37.16%. MS m / z: C465H597F8N146O289P42S8, [M+1]+, the theoretical value: 14787.45, the measured value: 14787.06.

[0953] 3.4.4.8 Synthesis of HJY-0203-DS010

[0954] Mix HJY-0203-SS with HJY-02-AS001-dp02 and HJY-03-AS001 in equimolar ratio in water for injection, heat to 70-95℃, then cool to room temperature, form a double-stranded structure by hydrogen bonding, obtain the final product HJY-0203-DS010.

[0955] 3.4.5 Synthesis of HJY-0203-DS011

[0956] 3.4.5.1 Synthesis of HJY-02-S001-MMT

[0957] Wherein, the sequence of the sense strand of siRNA1 is 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3'

[0958] The nucleotide monomers are sequentially connected one by one in the order of the above sequences from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 are adopted, including the reaction conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method, and the reaction is cycled for 22 times.

[0959] 3.4.5.2 Synthesis of HJY-03-S001-MMT:

[0960] wherein the sequence of the sense strand of siRNA2 is 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3'

[0961] The nucleotide monomers are sequentially connected one by one in the order of the above sequences from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 are adopted, including the reaction conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method, and the reaction is cycled for 22 times.

[0962] 3.4.5.3 Synthesis of HJY-02-AS001-dp02

[0963] The sequence of the antisense strand of siRNA1 is 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3'

[0964] The nucleotide monomers are sequentially connected one by one in the order of the above sequences from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 are adopted, including the reaction conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method. The reaction is cycled for 23 times.

[0965] 3.4.5.4 Synthesis of HJY-03-AS001-dp02

[0966] The sequence of the antisense strand of siRNA2 is 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3'

[0967] The nucleotide monomers are sequentially connected one by one in the order of the above sequences from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 are adopted, including the reaction conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method. The reaction is cycled for 23 times.

[0968] 3.4.5.5 Synthesis of HJY-0203-C2:

[0969] HJY-02-S001-MMT (0.4 g, 0.053 mmol) was dissolved in 2 mL of ultrapure water, mixed with 2 volumes of ice HOAc, and then placed in a 35 °C shaker for 1 h. After the reaction was completed, Na2CO3 was added, and the product was obtained as a white powder after drying. The yield was 81.51%. MS m / z: C225H297F3N85O135P21S4, [M+1]+, the theoretical value was 7178.99, and the measured value was 7178.26.

[0970] HJY-0203-C1 (0.31 g, 0.043 mmol) was dissolved in 0.1 M Na2B4O7 solution (1 mL), and then Azido-Aca-NHS (0.033 g, 0.129 mmol, azido-C5-succinimidyl ester) was dissolved in 1 mL of DMSO. The solution was transferred to the nucleic acid solution, and the reaction was carried out at 35 °C for 1 h. After the reaction was completed, NaOAc was added, and the product was obtained as a light yellow powder after drying. The yield was 79.11%. MS m / z: C231H306F3N88O136P21S4, [M+1]+, the theoretical value was 7327.15, and the measured value was 7326.84.

[0971] 3.4.5.6 Synthesis of HJY-0203-C4:

[0972] HJY-03-S001-MMT (0.4 g, 0.053 mmol) was dissolved in 2 mL of ultrapure water, mixed with 2 volumes of ice HOAc, and then placed in a 35 °C shaker for 1 h. After the reaction was completed, Na2CO3 was added, and the product was obtained as a white powder after drying. The yield was 86.78%. MS m / z: C225H296F3N84O135P21S4, [M+1]+, the theoretical value was 7172.98, and the measured value was 7172.32.

[0973] HJY-0203-C3 (0.33 g, 0.046 mmol) was dissolved in 0.1 M Na2B4O7 solution (1 mL), and then DBCO-NHS ester (0.055 g, 0.138 mmol, Dibenzocyclooctyne-active ester) was dissolved in 1 mL of DMSO. The solution was transferred to the nucleic acid solution, and the reaction was carried out at 35 °C for 1 h. After the reaction was completed, NaOAc was added, and the product was obtained as a light yellow powder after drying. The yield was 75.75%. MS m / z: C244H309F3N85O137P21S4, [M+1]+, the theoretical value was 7460.30, and the measured value was 7760.01.

[0974] 3.4.5.7 Synthesis of HJY-0203-SS

[0975] HJY-0203-C2 (0.2 g, 0.027 mmol) and HJY-0203-C4 (0.244 g, 0.033 mmol) were weighed and dissolved in 2 mL of ultrapure water. After stirring for 4 h, the control was qualified, and direct preparation purification was performed. Concentration to 1 mL, UV quantification, to obtain 151 mg of HJY-0203-SS, the yield was 37.16%. MS m / z: C465H597F8N146O289P42S8, [M+1]+, Theoretical: 14787.45, Found: 14787.06.

[0976] 3.4.5.8 Synthesis of HJY-0203-DS011

[0977] HJY-0203-SS was mixed with HJY-02-AS001 and HJY-03-AS001-dp02 in equimolar ratio in water for injection, heated to 70-95°C, then cooled to room temperature, and allowed to form a double-stranded structure by hydrogen bonding to obtain the final product HJY-0203-DS011.

[0978] 3.4.6 Synthesis of HJY-0203-DS012

[0979] 3.4.6.1 Synthesis of HJY-02-S001-MMT

[0980] wherein the sequence of the sense strand of siRNA1 is 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3'

[0981] According to the above sequence order, nucleotide monomers were connected one by one from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including deprotection, coupling, capping, and oxidation reaction conditions in solid-phase synthesis method, and the reaction was cycled for 22 times.

[0982] 3.4.6.2 Synthesis of HJY-03-S001-MMT

[0983] wherein the sequence of the sense strand of siRNA2 is 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3'

[0984] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid phase synthesis method. The reaction was cycled 22 times.

[0985] 3.4.6.3 Synthesis of HJY-02-AS001-dp01

[0986] The sequence of the antisense strand of siRNA1 is: 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3'

[0987] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid phase synthesis method. The reaction was cycled 23 times.

[0988] 3.4.6.4 Synthesis of HJY-03-AS001-dp01

[0989] The sequence of the antisense strand of siRNA2 is: 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3'

[0990] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid phase synthesis method. The reaction was cycled 23 times.

[0991] 3.4.6.5 Synthesis of HJY-0203-C2:

[0992] HJY-02-S001-MMT (0.4 g, 0.053 mmol) was dissolved in 2 mL of ultrapure water, 2 volumes of ice HOAc were added and mixed, then placed in a 35°C shaking bed for 1 h, the control was qualified, Na2CO3 was added, settled, dried to obtain 0.31 g of white powder, yield 81.51%. MS m / z: C225H297F3N85O135P21S4, [M+1]+, theoretical: 7178.99, found: 7178.26.

[0993] HJY-0203-C1 (0.31 g, 0.043 mmol) was weighed, 0.1 M Na2B4O7 solution (1 mL) was added, and it was completely dissolved; another azido-Aca-NHS (0.033 g, 0.129 mmol, azido-C5-succinimidyl ester) was dissolved in 1 mL DMSO, the solution was transferred to the nucleic acid solution, and it was placed on a 35°C shaker for 1 h of reaction. After the control was qualified, NaOAc was added, centrifuged, settled, and dried to obtain 0.25 g of light yellow powder, with a yield of 79.11%. MS m / z: C231H306F3N88O136P21S4, [M+1]+, theoretical: 7327.15, found: 7326.84.

[0994] 3.4.6.6 Synthesis of HJY-0203-C4

[0995] HJY-03-S001-MMT (0.4 g, 0.053 mmol) was weighed, 2 mL of ultrapure water was added to dissolve it, 2 volumes of ice HOAc were added, mixed, and then placed on a 35°C shaker for 1 h of reaction. After the control was qualified, Na2CO3 was added, settled, and dried to obtain 0.33 g of white powder, with a yield of 86.78%. MS m / z: C225H296F3N84O135P21S4, [M+1]+, theoretical: 7172.98, found: 7172.32.

[0996] HJY-0203-C3 (0.33 g, 0.046 mmol) was weighed, 0.1 M Na2B4O7 solution (1 mL) was added, and it was completely dissolved; another DBCO-NHS ester (0.055 g, 0.138 mmol, azido-dibenzocyclooctyne-active ester) was dissolved in 1 mL DMSO, the solution was transferred to the nucleic acid solution, and it was placed on a 35°C shaker for 1 h of reaction. After the control was qualified, NaOAc was added, centrifuged, settled, and dried to obtain 0.26 g of light yellow powder, with a yield of 75.75%. MS m / z: C244H309F3N85O137P21S4, [M+1]+, theoretical: 7460.30, found: 7760.01.

[0997] 3.4.6.7 Synthesis of HJY-0203-SS

[0998] HJY-0203-C2 (0.2 g, 0.027 mmol) and HJY-0203-C4 (0.244 g, 0.033 mmol) were weighed and dissolved in 2 mL of ultrapure water. After stirring for 4 h, the control was qualified, and direct preparation purification was performed. After concentration to 1 mL, UV quantification was performed to obtain 151 mg of HJY-0203-SS, with a yield of 37.16%. MS m / z: C465H597F8N146O289P42S8, [M+1]+, the theoretical value is 14787.45, and the measured value is 14787.06.

[0999] 3.4.6.8 Synthesis of HJY-0203-DS012

[1000] HJY-0203-SS was mixed with HJY-02-AS001-dp01 and HJY-03-AS001-dp01 in an equimolar ratio in water for injection and heated to 70-95°C, then cooled to room temperature, and allowed to form a double-stranded structure through hydrogen bonding to obtain the final product HJY-0203-DS012.

[1001] 3.4.7 Synthesis of HJY-0203-DS013

[1002] 3.4.7.1 Synthesis of HJY-02-S001-MMT

[1003] wherein the sequence of the sense strand of siRNA1 is 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3'

[1004] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method, and the cycle reaction was repeated for 22 times.

[1005] 3.4.7.2 Synthesis of HJY-03-S001-MMT:

[1006] wherein the sequence of the sense strand of siRNA2 is 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3'

[1007] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method, and the cycle reaction was repeated for 22 times.

[1008] 3.4.7.3 HJY-02-AS001-dp02

[1009] The sequence of the antisense strand of siRNA1 is: 5'UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3 '

[1010] The nucleotide monomers were sequentially connected from 3'-5' direction according to the sequence order by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were adopted, including the deprotection, coupling, capping, and oxidation reaction conditions in the solid-phase synthesis method. The reaction was cycled for 23 times.

[1011] 3.4.7.4 HJY-03-AS001-dp02

[1012] The sequence of the antisense strand of siRNA2 is: 5'UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3 '

[1013] The nucleotide monomers were sequentially connected from 3'-5' direction according to the sequence order by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were adopted, including the deprotection, coupling, capping, and oxidation reaction conditions in the solid-phase synthesis method. The reaction was cycled for 23 times.

[1014] 3.4.7.5 Synthesis of HJY-0203-C2:

[1015] HJY-02-S001-MMT (0.4 g, 0.053 mmol) was dissolved in 2 mL of ultrapure water, mixed with 2 volumes of ice HOAc, and placed in a 35°C shaking bed for 1 h. After the control was qualified, Na2CO3 was added, settled, dried to obtain 0.31 g of white powder, with a yield of 81.51%. MS m / z: C225H297F3N85O135P21S4, [M+1]+, theoretical: 7178.99, actual: 7178.26.

[1016] HJY-0203-C1 (0.31 g, 0.043 mmol) was weighed, 0.1 M Na2B4O7 solution (1 mL) was added, and completely dissolved; Azido-Aca-NHS (0.033 g, 0.129 mmol, azido-C5-succinimidyl ester) was dissolved in 1 mL of DMSO, and the solution was transferred to the nucleic acid solution, which was placed in a 35°C shaking bed for 1 h. After the control was qualified, NaOAc was added, centrifuged, settled, dried to obtain 0.25 g of light yellow powder, with a yield of 79.11%. MS m / z: C231H306F3N88O136P21S4, [M+1]+, theoretical: 7327.15, actual: 7326.84.

[1017] 3.4.7.6 Synthesis of HJY-0203-C4

[1018] HJY-03-S001-MMT (0.4 g, 0.053 mmol) was weighed into 2 mL of ultrapure water and dissolved. After mixing with 2 volumes of ice HOAc, it was placed in a 35 °C shaker for 1 h. After the control was qualified, Na2CO3 was added, settled, and dried to obtain 0.33 g of white powder with a yield of 86.78%. MS m / z: C225H296F3N84O135P21S4, [M+1]+, theoretical: 7172.98, found: 7172.32.

[1019] HJY-0203-C3 (0.33 g, 0.046 mmol) was weighed and 0.1 M Na2B4O7 solution (1 mL) was added to completely dissolve it. DBCO-NHS ester (0.055 g, 0.138 mmol, azadibenzocyclooctyne-active ester) was dissolved in 1 mL of DMSO, and the solution was transferred to the nucleic acid solution. It was placed in a 35 °C shaker for 1 h. After the control was qualified, NaOAc was added, centrifuged, settled, and dried to obtain 0.26 g of light yellow powder with a yield of 75.75%. MS m / z: C244H309F3N85O137P21S4, [M+1]+, theoretical: 7460.30, found: 7760.01.

[1020] 3.4.7.7 Synthesis of HJY-0203-SS

[1021] HJY-0203-C2 (0.2 g, 0.027 mmol) and HJY-0203-C4 (0.244 g, 0.033 mmol) were weighed and dissolved in 2 mL of ultrapure water. After stirring for 4 h, the control was qualified, and direct preparation purification was performed. After concentration to 1 mL, UV quantification was performed to obtain 151 mg of HJY-0203-SS with a yield of 37.16%. MS m / z: C465H597F8N146O289P42S8, [M+1]+, theoretical: 14787.45, found: 14787.06.

[1022] 3.4.7.8 Synthesis of HJY-0203-DS013

[1023] HJY-0203-SS was mixed with HJY-02-AS001-dp02 and HJY-03-AS001-dp02 in equimolar ratio in water for injection, heated to 70-95°C, then cooled at room temperature, to form a double-stranded structure by hydrogen bonding, to obtain the final product HJY-0203-DS013.

[1024] 3.4.8 Example 3.4-8 Synthesis of HJY-0203-DS014

[1025] 3.4.8.1 Synthesis of HJY-02-AS001-MMT

[1026] wherein the sequence of the sense strand of siRNA1 is 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3'

[1027] The nucleotide monomers were sequentially connected in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method, and the reaction was cycled for 24 times.

[1028] 3.4.8.2 Synthesis of HJY-03-AS001-MMT:

[1029] wherein the sequence of the sense strand of siRNA2 is 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3'

[1030] The nucleotide monomers were sequentially connected in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method, and the reaction was cycled for 24 times.

[1031] 3.4.8.3 HJY-02-S001-dp01

[1032] The sequence of the antisense strand of siRNA1 is 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3'

[1033] The nucleotide monomers were sequentially connected in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method. The reaction was cycled for 21 times.

[1034] 3.4.8.4 HJY-03-S001-dp02

[1035] Antisense strand sequence of siRNA2: 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3'

[1036] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were adopted, including the deprotection, coupling, capping, and oxidation reaction conditions in the solid-phase synthesis method. The reaction was cycled for 21 times.

[1037] 3.4.8.5 Synthesis of HJY-0203-C6:

[1038] HJY-03-AS001-MMT (0.5 g, 0.061 mmol) was weighed and dissolved in 2 mL of ultrapure water, 2 volumes of ice HOAc were added and mixed, then placed in a 35°C shaking bed for 1 h of reaction, the control was qualified, Na2CO3 was added, settled, dried to obtain 0.35 g of white powder, the yield was 73.36%. MS m / z: C244H322F1N80O158P23S4, [M+1]+, theoretical: 7763.29, found: 7762.99.

[1039] HJY-0203-C5 (0.35 g, 0.045 mmol) was weighed and added with 0.1 M Na2B4O7 solution (1 mL), completely dissolved; Azido-Aca-NHS (0.034 g, 0.135 mmol, azido-C5-succinimidyl ester) was dissolved in 1 mL of DMSO, and the solution was transferred to the nucleic acid solution, placed in a 35°C shaking bed for 1 h of reaction, the control was qualified, NaOAc was added, centrifuged, settled, dried to obtain 0.25 g of light yellow powder, the yield was 70.17%. MS m / z: C250H331F1N83O159P23S4, [M+1]+, theoretical: 7902.45, found: 7902.06.

[1040] 3.4.8.6 Synthesis of HJY-0203-C8:

[1041] HJY-02-AS001-MMT (0.5 g, 0.061 mmol) was weighed and dissolved in 2 mL of ultrapure water, 2 volumes of ice HOAc were added and mixed, then placed in a 35°C shaking bed for 1 h of reaction, the control was qualified, Na2CO3 was added, settled, dried to obtain 0.36 g of white powder, the yield was 75.44%. MS m / z: C244H322F1N80O160P23S4, [M+1]+, theoretical: 7795.29, found: 7794.86.

[1042] HJY-0203-C7 (0.36 g, 0.046 mmol) was weighed, 0.1 M Na2B4O7 solution (1 mL) was added, and it was completely dissolved; DBCO-NHS ester (0.056 g, 0.138 mmol, azadibenzocyclooctyne-active ester) was dissolved in 1 mL of DMSO, and the solution was transferred to the nucleic acid solution, and it was placed in a 35°C shaking bed for 1 h of reaction, and the control was qualified, NaOAc was added, centrifuged, settled, and dried to obtain 0.27 g of light yellow powder, with a yield of 72.33%. MS m / z: C263H335F1N81O162P23S4, [M+1]+, the theoretical value was 8082.61, and the measured value was 8082.05.

[1043] 3.4.8.7 Synthesis of HJY-0203-AS

[1044] HJY-0203-C6 (0.2 g, 0.025 mmol) and HJY-0203-C4 (0.245 g, 0.03 mmol) were weighed and dissolved in 2 mL of ultrapure water. After stirring for 4 h, the control was qualified, and direct preparation purification was performed. After concentration to 1 mL, UV quantification was performed to obtain 161 mg of HJY-0203-AS, with a yield of 39.55%. MS m / z: C513H666F2N164O321P46S8, [M+1]+, the theoretical value was 15985.06, and the measured value was 15984.69.

[1045] 3.4.8.8 Synthesis of HJY-0203-DS014

[1046] HJY-0203-AS was mixed with HJY-02-SS001-dp01 and HJY-03-SS001-dp02 in an equimolar ratio in water for injection, heated to 70-95°C, and then cooled to room temperature, so that a double-stranded structure was formed by hydrogen bonding to obtain the final product HJY-0203-DS014.

[1047] 3.4.9 Synthesis of HJY-0203-DS015

[1048] 3.4.9.1 Synthesis of HJY-02-AS001-MMT

[1049] wherein the sequence of the sense strand of siRNA1 is 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3'

[1050] The nucleotide monomers are sequentially connected one by one in the order of the above sequences from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 are adopted, including the conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method, and the reaction is cycled for 24 times.

[1051] 3.4.9.2 Synthesis of HJY-03-AS001-MMT:

[1052] wherein the sequence of the sense strand of siRNA2 is 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3'

[1053] The nucleotide monomers are sequentially connected one by one in the order of the above sequences from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 are adopted, including the conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method, and the reaction is cycled for 24 times.

[1054] 3.4.9.3 Synthesis of HJY-02-S001-dp02

[1055] The sequence of the antisense strand of siRNA1 is 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3'

[1056] The nucleotide monomers are sequentially connected one by one in the order of the above sequences from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 are adopted, including the conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method. The reaction is cycled for 21 times.

[1057] 3.4.9.4 Synthesis of HJY-03-S001-dp01

[1058] The sequence of the antisense strand of siRNA2 is 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3'

[1059] The nucleotide monomers are sequentially connected one by one in the order of the above sequences from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 are adopted, including the conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method. The reaction is cycled for 21 times.

[1060] 3.4.9.5 Synthesis of HJY-0203-C6:

[1061] HJY-03-AS001-MMT (0.5 g, 0.061 mmol) was dissolved in 2 mL of ultrapure water, mixed with 2 volumes of ice HOAc, and then placed in a 35 °C shaker for 1 h. After the reaction was completed, Na2CO3 was added, and the product was obtained as a white powder after drying. The yield was 73.36%. MS m / z: C244H322F1N80O158P23S4, [M+1]+, the theoretical value was 7763.29, and the measured value was 7762.99.

[1062] HJY-0203-C5 (0.35 g, 0.045 mmol) was dissolved in 0.1 M Na2B4O7 solution (1 mL), and then Azido-Aca-NHS (0.034 g, 0.135 mmol, azido-C5-succinimidyl ester) dissolved in 1 mL of DMSO was added to the nucleic acid solution. The solution was placed in a 35 °C shaker for 1 h. After the reaction was completed, NaOAc was added, and the product was obtained as a light yellow powder after drying. The yield was 70.17%. MS m / z: C250H331F1N83O159P23S4, [M+1]+, the theoretical value was 7902.45, and the measured value was 7902.06.

[1063] 3.4.9.6 Synthesis of HJY-0203-C8:

[1064] HJY-02-AS001-MMT (0.5 g, 0.061 mmol) was dissolved in 2 mL of ultrapure water, mixed with 2 volumes of ice HOAc, and then placed in a 35 °C shaker for 1 h. After the reaction was completed, Na2CO3 was added, and the product was obtained as a white powder after drying. The yield was 75.44%. MS m / z: C244H322F1N80O160P23S4, [M+1]+, the theoretical value was 7795.29, and the measured value was 7794.86.

[1065] HJY-0203-C7 (0.36 g, 0.046 mmol) was dissolved in 0.1 M Na2B4O7 solution (1 mL), and then DBCO-NHS ester (0.056 g, 0.138 mmol, Dibenzocyclooctyne-active ester) dissolved in 1 mL of DMSO was added to the nucleic acid solution. The solution was placed in a 35 °C shaker for 1 h. After the reaction was completed, NaOAc was added, and the product was obtained as a light yellow powder after drying. The yield was 72.33%. MS m / z: C263H335F1N81O162P23S4, [M+1]+, the theoretical value was 8082.61, and the measured value was 8082.05.

[1066] 3.4.9.7 Synthesis of HJY-0203-AS

[1067] HJY-0203-C6 (0.2 g, 0.025 mmol) and HJY-0203-C4 (0.245 g, 0.03 mmol) were weighed and dissolved in 2 mL of ultrapure water. After stirring for 4 h, the control was qualified, and direct preparation purification was performed. After concentration to 1 mL, UV quantification was performed to obtain 161 mg of HJY-0203-AS, with a yield of 39.55%. MS m / z: C513H666F2N164O321P46S8, [M+1]+, Theoretical: 15985.06, Found: 15984.69.

[1068] 3.4.9.8 Synthesis of HJY-0203-DS015

[1069] HJY-0203-AS was mixed with HJY-02-SS001-dp02 and HJY-03-SS001-dp01 in an equimolar ratio in water for injection and heated to 70-95°C, then cooled to room temperature, allowing it to form a double-stranded structure through hydrogen bonding to obtain the final product HJY-0203-DS015.

[1070] 3.4.10 Synthesis of HJY-0203-DS016

[1071] 3.4.10.1 Synthesis of HJY-02-AS001-MMT

[1072] wherein the sequence of the sense strand of siRNA1 is 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3'

[1073] The nucleotide monomers were connected one by one in the order of 3'-5' according to the above sequence by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in solid-phase synthesis, and the reaction was cycled for 24 times.

[1074] 3.4.10.2 Synthesis of HJY-03-AS001-MMT

[1075] wherein the sequence of the sense strand of siRNA2 is 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3'

[1076] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid phase synthesis method. The reaction was cycled for 24 times.

[1077] 3.4.10.3 HJY-02-S001-dp02

[1078] The antisense strand sequence of siRNA1 is: 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3'

[1079] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid phase synthesis method. The reaction was cycled for 24 times.

[1080] 3.4.10.4 HJY-03-S001

[1081] The antisense strand sequence of siRNA2 is: 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3'

[1082] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid phase synthesis method. The reaction was cycled for 24 times.

[1083] 3.4.10.5 Synthesis of HJY-0203-C6:

[1084] HJY-03-AS001-MMT (0.5 g, 0.061 mmol) was dissolved in 2 mL of ultrapure water, 2 volumes of ice HOAc were added and mixed, then placed in a 35°C shaking bed for 1 h, the control was qualified, Na2CO3 was added, settled, dried to obtain 0.35 g of white powder, the yield was 73.36%. MS m / z: C244H322F1N80O158P23S4, [M+1]+, theoretical: 7763.29, found: 7762.99.

[1085] HJY-0203-C5 (0.35 g, 0.045 mmol) was weighed, 0.1 M Na2B4O7 solution (1 mL) was added, and it was completely dissolved; another DBCO-NHS ester (0.056 g, 0.138 mmol, Dibenzocyclooctyne-active ester) was dissolved in 1 mL of DMSO, and the solution was transferred to the nucleic acid solution, and it was placed in a 35 °C shaking bed for 1 h of reaction, and the control was qualified, NaOAc was added, centrifuged, settled, and dried to obtain 0.27 g of light yellow powder, with a yield of 72.33%. MS m / z: C263H335F1N81O162P23S4, [M+1]+, the theoretical value was 8082.61, and the measured value was 8082.05.

[1086] 3.4.10.6 Synthesis of HJY-0203-C8

[1087] HJY-02-AS001-MMT (0.5 g, 0.061 mmol) was weighed, 2 mL of ultrapure water was added to dissolve it, 2 volumes of ice HOAc were added, mixed, and then placed in a 35 °C shaking bed for 1 h of reaction, and the control was qualified, Na2CO3 was added, settled, and dried to obtain 0.36 g of white powder, with a yield of 75.44%. MS m / z: C244H322F1N80O160P23S4, [M+1]+, the theoretical value was 7795.29, and the measured value was 7794.86.

[1088] HJY-0203-C7 (0.36 g, 0.046 mmol) was weighed, 0.1 M Na2B4O7 solution (1 mL) was added, and it was completely dissolved; another DBCO-NHS ester (0.056 g, 0.138 mmol, Dibenzocyclooctyne-active ester) was dissolved in 1 mL of DMSO, and the solution was transferred to the nucleic acid solution, and it was placed in a 35 °C shaking bed for 1 h of reaction, and the control was qualified, NaOAc was added, centrifuged, settled, and dried to obtain 0.27 g of light yellow powder, with a yield of 72.33%. MS m / z: C263H335F1N81O162P23S4, [M+1]+, the theoretical value was 8082.61, and the measured value was 8082.05.

[1089] 3.4.10.7 Synthesis of HJY-0203-AS

[1090] HJY-0203-C6 (0.2 g, 0.025 mmol) and HJY-0203-C4 (0.245 g, 0.03 mmol) were weighed and dissolved in 2 mL of ultrapure water. After stirring for 4 h, the control was qualified, and direct preparation purification was performed. After concentration to 1 mL, UV quantification was performed to obtain 161 mg of HJY-0203-AS, with a yield of 39.55%. MS m / z: C513H666F2N164O321P46S8, [M+1]+, Theoretical: 15985.06, Found: 15984.69.

[1091] 3.4.10.8 Synthesis of HJY-0203-DS016

[1092] HJY-0203-AS was mixed with HJY-02-SS001-dp02 and HJY-03-SS001 in an equimolar ratio in water for injection and heated to 70-95°C, then cooled to room temperature, allowing it to form a double-stranded structure through hydrogen bonding to obtain the final product HJY-0203-DS016.

[1093] 3.4.11 Synthesis of HJY-0203-DS017

[1094] 3.4.11.1 Synthesis of HJY-02-AS001-MMT

[1095] wherein the sequence of the sense strand of siRNA1 is 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3'

[1096] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method, and the cycle reaction was repeated for 24 times.

[1097] 3.4.11.2 Synthesis of HJY-03-AS001-MMT

[1098] wherein the sequence of the sense strand of siRNA2 is 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3'

[1099] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method, and the cycle reaction was repeated for 24 times.

[1100] 3.4.11.3 HJY-02-S001

[1101] Antisense sequence of siRNA1: 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3'

[1102] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid phase synthesis. The same synthesis conditions as described in 3.4.1.1 were adopted, including the conditions of deprotection, coupling, capping, and oxidation in the solid phase synthesis method. The reaction was cycled for 21 times.

[1103] 3.4.11.4 HJY-03-S001-dp02

[1104] Antisense sequence of siRNA2: 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3'

[1105] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid phase synthesis. The same synthesis conditions as described in 3.4.1.1 were adopted, including the conditions of deprotection, coupling, capping, and oxidation in the solid phase synthesis method. The reaction was cycled for 21 times.

[1106] 3.4.11.5 Synthesis of HJY-0203-C6:

[1107] HJY-03-AS001-MMT (0.5 g, 0.061 mmol) was weighed and dissolved in 2 mL of ultrapure water, mixed with 2 volumes of ice HOAc, and placed in a 35°C shaking bed for 1 h of reaction. After passing the control, Na2CO3 was added, settled, dried to obtain 0.35 g of white powder, with a yield of 73.36%. MS m / z: C244H322F1N80O158P23S4, [M+1]+, theoretical: 7763.29, actual: 7762.99.

[1108] HJY-0203-C5 (0.35 g, 0.045 mmol) was weighed and added to 0.1 M Na2B4O7 solution (1 mL) and completely dissolved. Azido-Aca-NHS (0.034 g, 0.135 mmol, azido-C5-succinimidyl ester) was dissolved in 1 mL of DMSO, and the solution was transferred to the nucleic acid solution, placed in a 35°C shaking bed for 1 h of reaction. After passing the control, NaOAc was added, centrifuged, settled, dried to obtain 0.25 g of light yellow powder, with a yield of 70.17%. MS m / z: C250H331F1N83O159P23S4, [M+1]+, theoretical: 7902.45, actual: 7902.06.

[1109] 3.4.11.6 Synthesis of HJY-0203-C8:

[1110] HJY-02-AS001-MMT (0.5 g, 0.061 mmol) was dissolved in 2 mL of ultrapure water, mixed with 2 volumes of ice HOAc, and placed in a 35 °C shaker for 1 h. After the control was qualified, Na2CO3 was added, and the product was obtained as a white powder after drying. The yield was 75.44%. MS m / z: C244H322F1N80O160P23S4, [M+1]+, the theoretical value: 7795.29, the actual value: 7794.86.

[1111] HJY-0203-C7 (0.36 g, 0.046 mmol) was dissolved in 0.1 M Na2B4O7 solution (1 mL), and DBCO-NHS ester (0.056 g, 0.138 mmol, azadibenzocyclooctyne-active ester) was dissolved in 1 mL of DMSO. The solution was transferred to the nucleic acid solution and placed in a 35 °C shaker for 1 h. After the control was qualified, NaOAc was added, and the product was obtained as a light yellow powder after centrifugation, sedimentation, and drying. The yield was 72.33%. MS m / z: C263H335F1N81O162P23S4, [M+1]+, the theoretical value: 8082.61, the actual value: 8082.05.

[1112] 3.4.11.7 Synthesis of HJY-0203-AS

[1113] HJY-0203-C6 (0.2 g, 0.025 mmol) and HJY-0203-C4 (0.245 g, 0.03 mmol) were dissolved in 2 mL of ultrapure water. After stirring for 4 h, the control was qualified, and the product was obtained after direct preparation and purification, UV quantification, and concentration to 1 mL. The yield of HJY-0203-AS was 161 mg, and the yield was 39.55%. MS m / z: C513H666F2N164O321P46S8, [M+1]+, the theoretical value: 15985.06, the actual value: 15984.69.

[1114] 3.4.11.8 Synthesis of HJY-0203-DS017

[1115] HJY-0203-AS, HJY-02-SS001, and HJY-03-SS001-dp02 were mixed in an equimolar ratio in water for injection and heated to 70-95 °C, followed by cooling to room temperature. The final product HJY-0203-DS017 was obtained by forming a double-stranded structure through hydrogen bonding.

[1116] 3.4.12 Example 3.4-12 Synthesis of HJY-0203-DS018

[1117] 3.4.12.1 Synthesis of HJY-02-AS001-MMT

[1118] wherein the sequence of the sense strand of siRNA1 is 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3'

[1119] The nucleotide monomers were connected one by one in the order of the sequence from 3'-5' by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method, and the reaction was cycled for 24 times.

[1120] 3.4.12.2 Synthesis of HJY-03-AS001-MMT:

[1121] wherein the sequence of the sense strand of siRNA2 is 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3'

[1122] The nucleotide monomers were connected one by one in the order of the sequence from 3'-5' by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method, and the reaction was cycled for 24 times.

[1123] 3.4.12.3 HJY-02-S001-dp01

[1124] The sequence of the antisense strand of siRNA1 is 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3'

[1125] The nucleotide monomers were connected one by one in the order of the sequence from 3'-5' by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method. The reaction was cycled for 21 times.

[1126] 3.4.12.4 HJY-03-S001-dp01

[1127] The sequence of the antisense strand of siRNA2 is 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3'

[1128] The nucleotide monomers were sequentially connected from 3'-5' direction according to the sequence order by the method of phosphoramidite solid-phase synthesis. The same synthetic conditions as described in 3.4.1.1 were used, including the deprotection, coupling, capping, and oxidation reaction conditions in the solid-phase synthesis method. The reaction was cycled for 21 times.

[1129] 3.4.12.5 Synthesis of HJY-0203-C6:

[1130] HJY-03-AS001-MMT (0.5 g, 0.061 mmol) was weighed and dissolved in 2 mL of ultrapure water. After mixing with 2 volumes of ice HOAc, it was placed in a 35°C shaking bed for 1 h of reaction. After passing the control, Na2CO3 was added, settled, and dried to obtain 0.35 g of white powder with a yield of 73.36%. MS m / z: C244H322F1N80O158P23S4, [M+1]+, theoretical: 7763.29, found: 7762.99.

[1131] HJY-0203-C5 (0.35 g, 0.045 mmol) was weighed and added to 0.1 M Na2B4O7 solution (1 mL) and completely dissolved. Azido-Aca-NHS (0.034 g, 0.135 mmol, azido-C5-succinimidyl ester) was dissolved in 1 mL of DMSO, and the solution was transferred to the nucleic acid solution. It was placed in a 35°C shaking bed for 1 h of reaction. After passing the control, NaOAc was added, centrifuged, settled, and dried to obtain 0.25 g of light yellow powder with a yield of 70.17%. MS m / z: C250H331F1N83O159P23S4, [M+1]+, theoretical: 7902.45, found: 7902.06.

[1132] 3.4.12.6 Synthesis of HJY-0203-C8:

[1133] HJY-02-AS001-MMT (0.5 g, 0.061 mmol) was weighed and dissolved in 2 mL of ultrapure water. After mixing with 2 volumes of ice HOAc, it was placed in a 35°C shaking bed for 1 h of reaction. After passing the control, Na2CO3 was added, settled, and dried to obtain 0.36 g of white powder with a yield of 75.44%. MS m / z: C244H322F1N80O160P23S4, [M+1]+, theoretical: 7795.29, found: 7794.86.

[1134] HJY-0203-C7 (0.36 g, 0.046 mmol) was weighed, 0.1 M Na2B4O7 solution (1 mL) was added, and it was completely dissolved; DBCO-NHS ester (0.056 g, 0.138 mmol, azadibenzocyclooctyne-active ester) was dissolved in 1 mL of DMSO, and the solution was transferred to the nucleic acid solution, and it was placed in a 35°C shaking bed for 1 h of reaction, and the control was qualified, NaOAc was added, centrifuged, settled, and dried to obtain 0.27 g of light yellow powder, with a yield of 72.33%. MS m / z: C263H335F1N81O162P23S4, [M+1]+, the theoretical value was 8082.61, and the measured value was 8082.05.

[1135] 3.4.12.7 Synthesis of HJY-0203-AS

[1136] HJY-0203-C6 (0.2 g, 0.025 mmol) and HJY-0203-C4 (0.245 g, 0.03 mmol) were weighed and dissolved in 2 mL of ultrapure water. After stirring for 4 h, the control was qualified, and direct preparation purification was performed. After concentration to 1 mL, UV quantification was performed to obtain 161 mg of HJY-0203-AS, with a yield of 39.55%. MS m / z: C513H666F2N164O321P46S8, [M+1]+, the theoretical value was 15985.06, and the measured value was 15984.69.

[1137] 3.4.12.8 Synthesis of HJY-0203-DS018

[1138] HJY-0203-AS was mixed with HJY-02-SS001-dp01 and HJY-03-sS001-dp01 in an equimolar ratio in water for injection, heated to 70-95°C, and then cooled to room temperature, so that a double-stranded structure was formed by hydrogen bonding to obtain the final product HJY-0203-DS018.

[1139] 3.4.13 Synthesis of HJY-0203-DS019

[1140] 3.4.13.1 Synthesis of HJY-02-AS001-MMT

[1141] wherein the sequence of the sense strand of siRNA1 is 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3'

[1142] The nucleotide monomers are connected one by one in the order of the above sequences from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 are adopted, including the reaction conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method, and the reaction is recycled for 24 times.

[1143] 3.4.13.2 Synthesis of HJY-03-AS001-MMT

[1144] The sequence of the sense strand of siRNA2 is 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3'

[1145] The nucleotide monomers are connected one by one in the order of the above sequences from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 are adopted, including the reaction conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method, and the reaction is recycled for 24 times.

[1146] 3.4.13.3 Synthesis of HJY-02-S001-dp02

[1147] The sequence of the antisense strand of siRNA1 is 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3'

[1148] The nucleotide monomers are connected one by one in the order of the above sequences from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 are adopted, including the reaction conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method. The reaction is recycled for 21 times.

[1149] 3.4.13.4 Synthesis of HJY-03-S001-dp02

[1150] The sequence of the antisense strand of siRNA2 is 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3'

[1151] The nucleotide monomers are connected one by one in the order of the above sequences from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 are adopted, including the reaction conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method. The reaction is recycled for 21 times.

[1152] 3.4.13.5 Synthesis of HJY-0203-C6

[1153] HJY-03-AS001-MMT (0.5 g, 0.061 mmol) was dissolved in 2 mL of ultrapure water, mixed with 2 volumes of ice HOAc, and then placed in a 35 °C shaker for 1 h. After the reaction was completed, Na2CO3 was added, and the product was obtained as a white powder after drying. The yield was 73.36%. MS m / z: C244H322F1N80O158P23S4, [M+1]+, the theoretical value was 7763.29, and the measured value was 7762.99.

[1154] HJY-0203-C5 (0.35 g, 0.045 mmol) was dissolved in 0.1 M Na2B4O7 solution (1 mL), and then Azido-Aca-NHS (0.034 g, 0.135 mmol, azido-C5-succinimidyl ester) dissolved in 1 mL of DMSO was added to the nucleic acid solution. The solution was placed in a 35 °C shaker for 1 h. After the reaction was completed, NaOAc was added, and the product was obtained as a light yellow powder after drying. The yield was 70.17%. MS m / z: C250H331F1N83O159P23S4, [M+1]+, the theoretical value was 7902.45, and the measured value was 7902.06.

[1155] 3.4.13.6 Synthesis of HJY-0203-C8:

[1156] HJY-02-AS001-MMT (0.5 g, 0.061 mmol) was dissolved in 2 mL of ultrapure water, mixed with 2 volumes of ice HOAc, and then placed in a 35 °C shaker for 1 h. After the reaction was completed, Na2CO3 was added, and the product was obtained as a white powder after drying. The yield was 75.44%. MS m / z: C244H322F1N80O160P23S4, [M+1]+, the theoretical value was 7795.29, and the measured value was 7794.86.

[1157] HJY-0203-C7 (0.36 g, 0.046 mmol) was dissolved in 0.1 M Na2B4O7 solution (1 mL), and then DBCO-NHS ester (0.056 g, 0.138 mmol, Dibenzocyclooctyne-active ester) dissolved in 1 mL of DMSO was added to the nucleic acid solution. The solution was placed in a 35 °C shaker for 1 h. After the reaction was completed, NaOAc was added, and the product was obtained as a light yellow powder after drying. The yield was 72.33%. MS m / z: C263H335F1N81O162P23S4, [M+1]+, the theoretical value was 8082.61, and the measured value was 8082.05.

[1158] 3.4.13.7 Synthesis of HJY-0203-AS

[1159] HJY-0203-C6 (0.2 g, 0.025 mmol) and HJY-0203-C4 (0.245 g, 0.03 mmol) were weighed and dissolved in 2 mL of ultrapure water. After stirring for 4 h, the control was qualified, and direct preparation purification was performed. After concentration to 1 mL, UV quantification was performed to obtain 161 mg of HJY-0203-AS, with a yield of 39.55%. MS m / z: C513H666F2N164O321P46S8, [M+1]+, Theoretical: 15985.06, Found: 15984.69.

[1160] 3.4.13.8 Synthesis of HJY-0203-DS019

[1161] HJY-0203-AS was mixed with HJY-02-SS001-dp02 and HJY-03-sS001-dp02 in an equimolar ratio in water for injection and heated to 70-95°C, then cooled to room temperature, allowing it to form a double-stranded structure through hydrogen bonding to obtain the final product HJY-0203-DS019.

[1162] 3.4.14 Synthesis of HJY-0203-DS020

[1163] 3.4.14.1 Synthesis of HJY-02-S001-MMT

[1164] wherein the sequence of the sense strand of siRNA1 is 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3'

[1165] The nucleotide monomers were connected one by one in the order of 3'-5' according to the above sequence by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in solid-phase synthesis, and the reaction was cycled for 22 times.

[1166] 3.4.14.2 Synthesis of HJY-03-S001-MMT

[1167] wherein the sequence of the sense strand of siRNA2 is 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3'

[1168] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid phase synthesis method. The reaction was cycled for 22 times.

[1169] 3.4.14.3 HJY-02-AS001-dp01

[1170] The antisense strand sequence of siRNA1 is: 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3'

[1171] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid phase synthesis method. The reaction was cycled for 23 times.

[1172] 3.4.14.4 HJY-03-AS001

[1173] The antisense strand sequence of siRNA2 is: 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3'

[1174] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid phase synthesis method. The reaction was cycled for 23 times.

[1175] 3.4.14.5 Synthesis of HJY-0203-C2:

[1176] HJY-02-S001-MMT (0.4 g, 0.053 mmol) was dissolved in 2 mL of ultrapure water, 2 volumes of ice HOAc were added and mixed, then placed in a 35°C shaking bed for 1 h, the control was qualified, Na2CO3 was added, settled, dried to obtain 0.31 g of white powder, the yield was 81.51%. MS m / z: C225H297F3N85O135P21S4, [M+1]+, theoretical: 7178.99, found: 7178.26.

[1177] HJY-0203-C1 (0.31 g, 0.043 mmol) was weighed, 0.1 M Na2B4O7 solution (1 mL) was added, and it was completely dissolved; another azido-Aca-NHS (0.033 g, 0.129 mmol, azido-C5-succinimidyl ester) was dissolved in 1 mL DMSO, the solution was transferred to the nucleic acid solution, and it was placed on a 35°C shaker for 1 h of reaction. After the control was qualified, NaOAc was added, centrifuged, settled, and dried to obtain 0.25 g of light yellow powder, with a yield of 79.11%. MS m / z: C231H306F3N88O136P21S4, [M+1]+, theoretical: 7327.15, found: 7326.84.

[1178] 3.4.14.6 Synthesis of HJY-0203-C4

[1179] HJY-03-S001-MMT (0.4 g, 0.053 mmol) was weighed, 2 mL of ultrapure water was added to dissolve it, 2 volumes of ice HOAc were added, mixed, and then placed on a 35°C shaker for 1 h of reaction. After the control was qualified, Na2CO3 was added, settled, and dried to obtain 0.33 g of white powder, with a yield of 86.78%. MS m / z: C225H296F3N84O135P21S4, [M+1]+, theoretical: 7172.98, found: 7172.32.

[1180] HJY-0203-C3 (0.33 g, 0.046 mmol) was weighed, 0.1 M Na2B4O7 solution (1 mL) was added, and it was completely dissolved; another DBCO-NHS ester (0.055 g, 0.138 mmol, azido-dibenzocyclooctyne-active ester) was dissolved in 1 mL DMSO, the solution was transferred to the nucleic acid solution, and it was placed on a 35°C shaker for 1 h of reaction. After the control was qualified, NaOAc was added, centrifuged, settled, and dried to obtain 0.26 g of light yellow powder, with a yield of 75.75%. MS m / z: C244H309F3N85O137P21S4, [M+1]+, theoretical: 7460.30, found: 7760.01.

[1181] 3.4.4.7 Synthesis of HJY-0203-SS

[1182] HJY-0203-C2 (0.2 g, 0.027 mmol) and HJY-0203-C4 (0.244 g, 0.033 mmol) were weighed and dissolved in 2 mL of ultrapure water. After stirring for 4 h, the control was qualified, and direct preparation purification was performed. After concentration to 1 mL, UV quantification was performed to obtain 151 mg of HJY-0203-SS, with a yield of 37.16%. MS m / z: C465H597F8N146O289P42S8, [M+1]+, the theoretical value is 14787.45, and the measured value is 14787.06.

[1183] 3.4.14.8 Synthesis of HJY-0203-DS020

[1184] HJY-0203-SS was mixed with HJY-02-AS001-dp01 and HJY-03-AS001 in an equimolar ratio in water for injection and heated to 70-95°C, and then cooled to room temperature. A double-stranded structure was formed by hydrogen bonding to obtain the final product HJY-0203-DS020.

[1185] 3.4.15 Synthesis of HJY-0203-DS021

[1186] 3.4.15.1 Synthesis of HJY-02-S001-MMT

[1187] wherein the sequence of the sense strand of siRNA1 is 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3'

[1188] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method, and the cycle reaction was repeated for 22 times.

[1189] 3.4.15.2 Synthesis of HJY-03-S001-MMT:

[1190] wherein the sequence of the sense strand of siRNA2 is 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3'

[1191] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method, and the cycle reaction was repeated for 22 times.

[1192] 3.4.15.3 HJY-02-AS001

[1193] Antisense strand sequence of siRNA1: 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3'

[1194] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were adopted, including the deprotection, coupling, capping, and oxidation reaction conditions in the solid-phase synthesis method. The reaction was cycled for 23 times.

[1195] 3.4.15.4 HJY-03-AS001-dp02

[1196] Antisense strand sequence of siRNA2: 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3'

[1197] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were adopted, including the deprotection, coupling, capping, and oxidation reaction conditions in the solid-phase synthesis method. The reaction was cycled for 23 times.

[1198] 3.4.15.5 Synthesis of HJY-0203-C2:

[1199] HJY-02-S001-MMT (0.4 g, 0.053 mmol) was dissolved in 2 mL of ultrapure water, mixed with 2 volumes of ice HOAc, and placed in a 35°C shaking bed for 1 h of reaction. After passing the control, Na2CO3 was added, settled, dried to obtain 0.31 g of white powder, with a yield of 81.51%. MS m / z: C225H297F3N85O135P21S4, [M+1]+, theoretical: 7178.99, actual: 7178.26.

[1200] HJY-0203-C1 (0.31 g, 0.043 mmol) was weighed, 0.1 M Na2B4O7 solution (1 mL) was added, and completely dissolved; Azido-Aca-NHS (0.033 g, 0.129 mmol, azido-C5-succinimidyl ester) was dissolved in 1 mL of DMSO, and the solution was transferred to the nucleic acid solution, placed in a 35°C shaking bed for 1 h of reaction. After passing the control, NaOAc was added, centrifuged, settled, dried to obtain 0.25 g of light yellow powder, with a yield of 79.11%. MS m / z: C231H306F3N88O136P21S4, [M+1]+, theoretical: 7327.15, actual: 7326.84.

[1201] 3.4.15.6 Synthesis of HJY-0203-C4

[1202] HJY-03-S001-MMT (0.4 g, 0.053 mmol) was weighed into 2 mL of ultrapure water and dissolved, 2 volumes of ice HOAc were added and mixed, then placed in a 35 °C shaker for 1 h, the control was qualified, Na2CO3 was added, settled, dried to obtain 0.33 g of white powder, yield 86.78%. MS m / z: C225H296F3N84O135P21S4, [M+1]+, theoretical: 7172.98, found: 7172.32.

[1203] HJY-0203-C3 (0.33 g, 0.046 mmol) was weighed, 0.1 M Na2B4O7 solution (1 mL) was added and completely dissolved; DBCO-NHS ester (0.055 g, 0.138 mmol, azadibenzocyclooctyne-active ester) was dissolved in 1 mL of DMSO, the solution was transferred to the nucleic acid solution, and placed in a 35 °C shaker for 1 h, the control was qualified, NaOAc was added, centrifuged, settled, dried to obtain 0.26 g of light yellow powder, yield 75.75%. MS m / z: C244H309F3N85O137P21S4, [M+1]+, theoretical: 7460.30, found: 7760.01.

[1204] 3.4.15.7 Synthesis of HJY-0203-SS

[1205] HJY-0203-C2 (0.2 g, 0.027 mmol) and HJY-0203-C4 (0.244 g, 0.033 mmol) were weighed, and 2 mL of ultrapure water was added to dissolve. Stirring for 4 h, the control was qualified, directly prepared and purified, concentrated to 1 mL, UV quantification, obtained 151 mg of HJY-0203-SS, yield 37.16%. MS m / z: C465H597F8N146O289P42S8, [M+1]+, theoretical: 14787.45, found: 14787.06.

[1206] 3.4.15.8 Synthesis of HJY-0203-DS021

[1207] HJY-0203-SS and HJY-02-AS001 and HJY-03-AS001-dp01 are mixed in equimolar ratio in water for injection, heated to 70-95°C, then cooled to room temperature, and allowed to form double-stranded structure by hydrogen bonding to obtain the final product HJY-0203-DS021, HJY-0203-DS022, HJY-0203-DS022, HJY-0203-DS024. The synthesis method is also the same, and the mass spectrum is shown in Figures 5, 6, 7, respectively. As long as the orthogonal reaction group and the connecting chain structure are connected to the 5' end of the sense strand, and the carrier structure is connected to the 3' end of the antisense strand, the inhibitor can be synthesized according to the synthesis method of the present application.

[1208] 3.4.16 Synthesis process of example 3.4-16, 17 DDP02, DDP03:

[1209] 3.4.16.1 Synthesis process of DDP02:

[1210] DDP02-DS002~DDP02-DS013 can be obtained according to this synthesis process, and as an example, the mass spectrum of DDP02-DS006 is shown in Figure 3.

[1211] 3.4.16.2 Synthesis process of DDP03:

[1212] As an example, the mass spectrum of DDP03-DS001 is shown in Figure 4.

[1213] 3.4.17 Example 3.4-18 HJY-0203-DS027 synthesis

[1214] 3.4.17.1 Synthesis of HJY-02-S001-MMT

[1215] wherein the sense strand sequence of siRNA1 is 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3'

[1216] The nucleotide monomers are connected in the order of 3'-5' direction according to the above sequence by the method of phosphoramidite solid phase synthesis. The same synthesis conditions as described in 3.4.1.1 are used, including the conditions of deprotection, coupling, capping and oxidation in solid phase synthesis method, and the reaction is cycled for 22 times.

[1217] 3.4.17.2 Synthesis of HJY-03-S001-MMT-HJYNSdp02:

[1218] The sequence of the sense strand of siRNA2 is 5' UsGsCAAAUAfGfUfCUACAAACCsAsA 3'

[1219] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method. The reaction was cycled for 22 times.

[1220] 3.4.17.3 HJY-02-AS001

[1221] The sequence of the antisense strand of siRNA1 is 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3'

[1222] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method. The reaction was cycled for 23 times.

[1223] 3.4.17.4 HJY-03-AS001

[1224] The sequence of the antisense strand of siRNA2 is 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3'

[1225] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in the solid-phase synthesis method. The reaction was cycled for 23 times.

[1226] 3.4.17.5 Synthesis of HJY-0203-C2:

[1227] HJY-02-S001-MMT (0.4 g, 0.053 mmol) was dissolved in 2 mL of ultrapure water, 2 volumes of ice HOAc were added and mixed, and then placed in a 35°C shaking bed for 1 h. After the control was qualified, Na2CO3 was added, settled, and dried to obtain 0.31 g of white powder, with a yield of 81.51%. MS m / z: C225H297F3N85O135P21S4, [M+1]+, theoretical: 7178.99, found: 7178.26.

[1228] HJY-0203-C1 (0.31 g, 0.043 mmol) was weighed, 0.1 M Na2B4O7 solution (1 mL) was added, and it was completely dissolved; another DBCO-NHS ester (0.055 g, 0.138 mmol, Dibenzocyclooctyne-active ester) was dissolved in 1 mL of DMSO, and the solution was transferred to the nucleic acid solution, and it was placed in a 35 °C shaking bed for 1 h. After the control was qualified, NaOAc was added, centrifuged, settled, and dried to obtain 0.26 g of light yellow powder, with a yield of 75.75%. MS m / z: C292H402F3N89O145P22S4, [M+1]+, the theoretical value: 8345.56, the measured value: 8345.11.

[1229] 3.4.17.6 Synthesis of HJY-0203-C10

[1230] HJY-03-S001-MMT-HJYNSdp02 (0.4 g, 0.048 mmol) was weighed, 2 mL of ultrapure water was added to dissolve it, 2 volumes of ice HOAc were added, mixed, and then placed in a 35 °C shaking bed for 1 h. After the control was qualified, Na2CO3 was added, settled, and dried to obtain 0.32 g of white powder, with a yield of 82.26%. MS m / z: C273H389F3N88O143P22S4, [M+1]+, the theoretical value: 8058.24, the measured value: 8057.85.

[1231] HJY-0203-C9 (0.32 g, 0.040 mmol) was weighed, 0.1 M Na2B4O7 solution (1 mL) was added, and it was completely dissolved; another DBCO-NHS ester (0.055 g, 0.138 mmol, Dibenzocyclooctyne-active ester) was dissolved in 1 mL of DMSO, and the solution was transferred to the nucleic acid solution, and it was placed in a 35 °C shaking bed for 1 h. After the control was qualified, NaOAc was added, centrifuged, settled, and dried to obtain 0.26 g of light yellow powder, with a yield of 75.75%. MS m / z: C292H402F3N89O145P22S4, [M+1]+, the theoretical value: 8345.56, the measured value: 8345.11.

[1232] 3.4.17.7 Synthesis of HJY-0203-SS1

[1233] HJY-0203-C2 (0.2 g, 0.027 mmol) and HJY-0203-C10 (0.275 g, 0.033 mmol) were weighed and dissolved in 2 mL of ultrapure water. After stirring for 4 h, the control was qualified, and direct preparation purification was performed. After concentration to 1 mL, UV quantification was performed to obtain 144 mg of HJY-0203-SS1, with a yield of 33.66%. MS m / z: C523H708F6N177O281P43S8, [M+1]+, theoretical: 15672.71, found: 15672.06.

[1234] 3.4.17.8 Synthesis of HJY-0203-DS027

[1235] HJY-0203-SS1 was mixed with HJY-02-AS001 and HJY-03-AS001 in an equimolar ratio in water for injection and heated to 70-95°C, then cooled to room temperature, to form a double-stranded structure by hydrogen bonding, to obtain the final product HJY-0203-DS027, and the mass spectrum is shown in Figure 10. HJY-0203-DS026 or other orthogonal reaction groups and linker structures connect the 5' end of the positive sense strand of the double-stranded RNA, and the synthesis process of the inhibitor connected to the 3' end of the positive sense strand is also the same, and the mass spectrum of HJY-0203-DS026 is shown in Figure 9.

[1236] 3.4.18 Synthesis of HJY-0203-DS025

[1237] 3.4.18.1 Synthesis of HJY-02-S001-MMT

[1238] wherein the sequence of the positive sense strand of siRNA1 is 5' GsGsCUACGAfAfAfAUCCAACCUsAsA 3'

[1239] The nucleotide monomers were connected one by one in the order of 3'-5' according to the above sequence by the method of phosphoramidite solid-phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the conditions of deprotection, coupling, capping, and oxidation in solid-phase synthesis, and the reaction was cycled for 22 times.

[1240] 3.4.18.2 Synthesis of HJY-03-S001-MMT:

[1241] wherein the sequence of the positive sense strand of siRNA2 is 5' UsGsCAA(Ahd)UAfGfUfCUACAAACCsAsA 3', and Ahd refers to a C coupled to the 2' position of an adenine ribonucleotide 16H 33 .

[1242] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the reaction conditions of deprotection, coupling, capping, and oxidation in the solid phase synthesis method. The reaction was cycled for 22 times.

[1243] 3.4.18.3 HJY-02-AS001

[1244] The antisense strand sequence of siRNA1: 5' UsdTsAGdGUdTGGAUdTUfUCGUAGCCsGsU 3'

[1245] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the reaction conditions of deprotection, coupling, capping, and oxidation in the solid phase synthesis method. The reaction was cycled for 23 times.

[1246] 3.4.18.4 HJY-03-AS001

[1247] The antisense strand sequence of siRNA2: 5' UsdTsGGdTUdTGUAGdACfUAUUUGCAsCsA 3'

[1248] The nucleotide monomers were connected one by one in the order of the above sequence from 3'-5' direction by the method of phosphoramidite solid phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the reaction conditions of deprotection, coupling, capping, and oxidation in the solid phase synthesis method. The reaction was cycled for 23 times.

[1249] 3.4.18.5 Synthesis of HJY-02-03-C2:

[1250] HJY-02-S001-MMT (0.4 g, 0.053 mmol) was dissolved in 2 mL of ultrapure water, 2 volumes of ice HOAc were added and mixed, then placed in a 35°C shaking bed for 1 h, the control was qualified, Na2CO3 was added, settled, dried to obtain 0.31 g of white powder, the yield was 81.51%. MS m / z: C225H297F3N85O135P21S4, [M+1]+, theoretical: 7178.99, found: 7178.26.

[1251] HJY-0203-C1 (0.31 g, 0.043 mmol) was weighed, 0.1 M Na2B4O7 solution (1 mL) was added, and it was completely dissolved; another DBCO-NHS ester (0.055 g, 0.138 mmol, Dibenzocyclooctyne-active ester) was dissolved in 1 mL of DMSO, and the solution was transferred to the nucleic acid solution, and it was placed in a 35°C shaking bed for 1 h. After the control was qualified, NaOAc was added, centrifuged, settled, and dried to obtain 0.27 g of light yellow powder, with a yield of 81.32%. MS m / z: C259H339F3N85O137P21S4, [M+1]+, the theoretical value was 7670.7, and the measured value was 7670.11.

[1252] 3.4.18.6 Synthesis of HJY-0203-C12

[1253] HJY-03-S001-MMT (0.4 g, 0.053 mmol) was weighed, 2 mL of ultrapure water was added to dissolve it, 2 volumes of ice HOAc were added, mixed, and then placed in a 35°C shaking bed for 1 h. After the control was qualified, Na2CO3 was added, settled, and dried to obtain 0.32 g of white powder, with a yield of 82.47%. MS m / z: C240H326F3N84O135P21S4, [M+1]+, the theoretical value was 7383.38, and the measured value was 7382.95.

[1254] HJY-0203-C11 (0.32 g, 0.043 mmol) was weighed, 0.1 M Na2B4O7 solution (1 mL) was added, and it was completely dissolved; another DBCO-NHS ester (0.055 g, 0.138 mmol, Dibenzocyclooctyne-active ester) was dissolved in 1 mL of DMSO, and the solution was transferred to the nucleic acid solution, and it was placed in a 35°C shaking bed for 1 h. After the control was qualified, NaOAc was added, centrifuged, settled, and dried to obtain 0.27 g of light yellow powder, with a yield of 81.32%. MS m / z: C259H339F3N85O137P21S4, [M+1]+, the theoretical value was 7670.7, and the measured value was 7670.11.

[1255] 3.4.18.7 Synthesis of HJY-0203-SS2

[1256] HJY-0203-C2 (0.2 g, 0.027 mmol) and HJY-0203-C12 (0.253 g, 0.033 mmol) were weighed and dissolved in 2 mL of ultrapure water. After stirring for 4 h, the control was qualified, and direct preparation purification was performed. After concentration to 1 mL, UV quantification was performed to obtain 164 mg of HJY-0203-SS2, with a yield of 40.49%. MS m / z: C490H645F6N173O273P42S8, [M+1]+, Theoretical: 14997.85, Found: 14997.33.

[1257] 3.4.18.8 Synthesis of HJY-0203-DS025

[1258] HJY-0203-SS2 was mixed with HJY-02-AS001 and HJY-03-AS001 in equimolar ratio in water for injection and heated to 70-95°C, then cooled to room temperature, to form a double-stranded structure by hydrogen bonding, to obtain the final product HJY-0203-DS025, and the mass spectrum is shown in Figure 8. Other orthogonal reaction groups and linkage structures connect the 5' end of the positive sense strand of the two double-stranded RNAs, and the synthesis process of the inhibitor coupled to the 2' position of a certain nucleotide in the positive sense strand is similar.

[1259] 3.4.19 Synthesis of DDP08-DS00502

[1260] 3.4.19.1 Synthesis of Klyo-08-S00103-MMT

[1261] wherein the sequence of the positive sense strand of siRNA1 is 5' CsUsAGACfCUfGUdTUUGCUUUUsGsU-3' MVIP09

[1262] The nucleotide monomers were connected one by one in the order of 3'-5' according to the above sequence by the method of phosphoramidite solid-phase synthesis. Each connection of a nucleotide monomer includes deprotection, coupling, capping, and oxidation reactions, cyclic reactions, and the same synthesis conditions as described in 3.4.1.1.

[1263] 3.4.19.2 Synthesis of Klyo-11-S00103-MMT

[1264] wherein the sequence of the positive sense strand of siRNA2 is 5' CsAsGCUCCUfUfAfUUGUUAUACsGsA-3' MVIP09

[1265] The nucleotide monomers were sequentially connected one by one in the order of the above sequences from 3'-5' direction by the method of phosphoramidite solid phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the deprotection, coupling, capping, and oxidation reaction conditions in the solid phase synthesis method. The reaction was cycled for 22 times.

[1266] 3.4.19.3 DDP08-AS00502-siRNA1 (3'-asRNA1-5')

[1267] The sequence of the antisense strand of siRNA1 is: 5' VPAsfCsAfAfAfAGfCAfAAfACfAGfGUfCUAGsAsAdTdT 3'

[1268] The nucleotide monomers were sequentially connected one by one in the order of the above sequences from 3'-5' direction by the method of phosphoramidite solid phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the deprotection, coupling, capping, and oxidation reaction conditions in the solid phase synthesis method. The reaction was cycled for 22 times.

[1269] 3.4.19.4 DDP08-AS00502-siRNA2 (3'-asRNA2-5')

[1270] The sequence of the antisense strand of siRNA1 is: 5' VPUsfCsGfUAfUAACAAfUAfAGfGAfGCsfUsGdTdT 3'

[1271] The nucleotide monomers were sequentially connected one by one in the order of the above sequences from 3'-5' direction by the method of phosphoramidite solid phase synthesis. The same synthesis conditions as described in 3.4.1.1 were used, including the deprotection, coupling, capping, and oxidation reaction conditions in the solid phase synthesis method. The reaction was cycled for 22 times.

[1272] 3.4.19.5 Synthesis of Klyo-08-S00103-C2

[1273] Dissolve Kylo-08-S00103-MMT in ultrapure water, mix with 2 volumes of ice HOAc, and place on a 35°C shaking table for 1 h of reaction. After passing the control, add Na2CO3, settle, and dry to obtain a powder.

[1274] Weigh Kylo-08-S00103-C1 and add 0.1 M Na2B4O7 solution to completely dissolve it. Dissolve Azido-Aca-NHS (azido-C5-succinimidyl ester) in DMSO, transfer the solution to the nucleic acid solution, and place it on a 35°C shaking table for 1 h of reaction. After passing the control, add NaOAc, centrifuge, settle, and dry to obtain a powder.

[1275] 3.4.19.6 Synthesis of Klyo-11-S00103-C4

[1276] Klyo-11-S00103-MMT was dissolved in ultrapure water, 2 volumes of ice HOAc were added, mixed well, and then placed in a 35 °C shaker for 1 h. After the control was qualified, Na2CO3 was added, settled, and dried to obtain a powder.

[1277] Klyo-11-S00103-C3 was weighed and added to a 0.1 M Na2B4O7 solution and completely dissolved. Azido-Aca-NHS (azido-C5-succinimidyl ester) was dissolved in 1 mL DMSO, and the solution was transferred to the nucleic acid solution. It was placed in a 35 °C shaker for 1 h. After the control was qualified, NaOAc was added, centrifuged, settled, and dried to obtain a powder.

[1278] 3.4.19.7 Synthesis of DDP08-Kylo-08-S00103-DBCO

[1279] Linker 8 was dissolved in DMF, and Kylo-08-S00103-C2 was dissolved in pure water. The linker 10 solution was added dropwise under stirring, and stirred for 0.5 h. After the control was qualified, it was directly prepared and purified, concentrated to 2 mL, and quantified by UV to obtain Kylo-08-S00103-DBCO.

[1280] 3.4.19.8 Synthesis of DDP08-S00502

[1281] DDP-Kylo-08-S00103-DBCO and Kylo-11-S00103-C4 were weighed and dissolved in ultrapure water. After stirring for 1 h, the control was qualified, and it was directly prepared and purified, concentrated, and quantified by UV to obtain DDP08-S00502.

[1282] 3.4.19.9 Synthesis of DDP08-DS00502

[1283] DDP08-S00502 was mixed with DDP08-AS00502-siRNA1 (3'-asRNA1-5') and DDP08-AS00502-siRNA2 (3'-asRNA2-5') in equimolar ratio in water for injection, heated to 70-95 °C, and then cooled to room temperature. It was allowed to form a double-stranded structure through hydrogen bonding to obtain the final product DDP08-DS00502.

[1284] The synthesis process of DDP09, DDP10 and DDP11 series compounds can refer to the synthesis process of DDP08 compound described above, mainly involving the replacement of linker9, linker10 and linker11.

[1285] In some embodiments, the antisense strand of the RNA inhibitor described herein is a sequence having at least 15 contiguous nucleotides identical to an antisense strand in Table 1, or a sequence differing by one, two or three nucleotides from an antisense strand in Table 1.

[1286] In some embodiments, the antisense strand of the RNA inhibitor described herein is a sequence having at least 15 contiguous nucleotides identical to an antisense strand in Table 1, or a sequence differing by one, two or three nucleotides from an antisense strand in Table 1.

[1287] In some embodiments, the double-stranded RNA inhibitor of the present application can be optionally conjugated with one or more ligands, which can be applied to the double-stranded RNA inhibitor of the present application as long as it can enhance the activity, cellular distribution or cellular uptake (e.g., into cells) of the double-stranded RNA inhibitor. The ligand can be connected to the sense strand, the antisense strand or both strands at the 3' end, the 5' end or both ends. The carrier is not limited to the MVIP listed in the present application, and can also include, but is not limited to: GalNAc carrier of any structure, cationic lipid carrier, viral carrier, lipophilic moiety, amphiphilic moiety, targeting group, small molecule drug, protein, peptide, antibody.

[1288] Part IV, in vitro cell verification experiment:

[1289] 4.1 The RNA inhibitor of this example is selected from Table 1, and the inhibitory effect of the DDP series RNA inhibitor is verified by free uptake of primary liver cells of cynomolgus monkeys.

[1290] Verification method: Freshly resuscitated primary cynomolgus monkey liver cells were prepared according to 4x10 4Cells were plated in 96-well plates coated with collagen, and after 3 h of cell adhesion, the diluted siRNA samples were added to treat the cells, which were then incubated in a 5% CO2, 37°C incubator. After 48 h of treatment, total RNA was extracted using the EZ-Mag Automated Trace Cells RNA Purification Kit PLUS kit (Suzhou Eppendorf Biomedicine Technology Co., Ltd., Catalog No. EZ4117), cDNA was prepared using the 4x EZscript Reverse Transcription Mix II (with gDNA Remover) kit (Suzhou Eppendorf Biomedicine Technology Co., Ltd., Catalog No. EZB-RT2GQ), and qPCR detection was performed using the Multiplex Probe qPCR Master Mix (Suzhou Eppendorf Biomedicine Technology Co., Ltd., Catalog No. EZ3304-R2).

[1291] Data analysis: The mRNA expression level of the target gene in each sample was calculated based on the Ct value, and the relative quantification method was used for calculation. The relative expression of the target gene was calculated using 2 -ΔΔCT .

[1292] The calculation formula is as follows: ΔCT = average Ct value of target gene - average Ct value of internal reference gene; ΔΔCT = ΔCT (treatment group) - ΔCT (control group);

[1293] The relative expression of the target gene mRNA = 2 -ΔΔCT .

[1294] The inhibition rate of the target gene = (1 - value of sample / mean value of control) x 100%, and the results are expressed as mean ± SD of three replicate wells. The mRNA inhibition effect of the RNA inhibitors at concentrations of 1000 nM, 100 nM, and 10 nM on C5, C3, and CFB genes in primary cynomolgus monkey hepatocytes was investigated. The test results are shown in Tables 2-7.

[1295] Table 4 Inhibition effect of DDP02 series RNA inhibitors on C5 and C3 mRNA in primary cynomolgus monkey hepatocytes

[1296] Table 5 Inhibition effect of DDP series RNA inhibitors on C5 and C3 mRNA in primary cynomolgus monkey hepatocytes

[1297] From Table 4, 5, it can be seen that, from the delivery effect of the multi-target inhibitor and the inhibitor mixture, it can be seen that the multi-target inhibitor (DDP02, DDP03, DDP04) of the application does not interfere with the inhibition effect of siRNA, and even shows a better trend than the delivery effect of the inhibitor mixture. DDP04-DS001 and DDP02-DS006 have the same sequence, only the linker chemical structure is different, from the comparison of the experimental data of the two, it can be seen that the delivery effect of DDP04 is better than that of DDP02.

[1298] Table 6 Inhibition effect of DDP04-DS003 series RNA inhibitors on PCSK9, AGT mRNA in primary cynomolgus monkey hepatocytes

[1299] From Table 6, it can be seen that, from the delivery effect of the multi-target inhibitor and the inhibitor mixture, it can be seen that the multi-target inhibitor DDP04 of the application does not interfere with the inhibition effect of siRNA, and even shows a better trend than the delivery effect of the inhibitor mixture.

[1300] Table 7 Inhibition effect of DDP04-DS004 series RNA inhibitors on PCSK9, APOC3 mRNA in primary cynomolgus monkey hepatocytes

[1301] From Table 7, it can be seen that, from the delivery effect of the multi-target inhibitor and the inhibitor mixture, it can be seen that the multi-target inhibitor DDP04 of the application does not interfere with the inhibition effect of siRNA, and even shows a better trend than the delivery effect of the inhibitor mixture.

[1302] Table 8 Inhibition effect of DDP04-DS005 series RNA inhibitors on PCSK9, LPA mRNA in primary cynomolgus monkey hepatocytes

[1303] From Table 8, it can be seen that, from the delivery effect of the multi-target inhibitor and the inhibitor mixture, it can be seen that the multi-target inhibitor DDP04 of the application does not interfere with the inhibition effect of siRNA, and even shows a better trend than the delivery effect of the inhibitor mixture.

[1304] Table 9 Inhibition effect of DDP04-DS006 series RNA inhibitors on PCSK9, ANGPTL3 mRNA in primary cynomolgus monkey hepatocytes

[1305] As can be seen from Table 9, compared with the delivery effect of the multi-target inhibitor and the inhibitor mixture, it can be seen that the multi-target inhibitor DDP04 of the present application does not interfere with the inhibition effect of siRNA, and even shows a better trend than the delivery effect of the inhibitor mixture.

[1306] Table 10 Inhibition effect of DDP04-DS006 series RNA inhibitors on PCSK9, ANGPTL3 mRNA in primary cynomolgus monkey hepatocytes

[1307] The siRNA sequence modification and the structure of the connecting carrier in DDP04-DS006 and DDP04-DS00601 are the same, and only the connection order of the orthogonal reaction group is different. As can be seen from Table 10, the multi-target inhibitor DDP04-DS006 and DDP04-DS00601 of the present application both have obvious inhibition effect, which shows that different connection orders have obvious effect.

[1308] The siRNA sequence modification and the structure of the connecting carrier in DDP04-DS006 and DDP04-DS00601 are the same, and only the connection order of the orthogonal reaction group is different. As can be seen from Table 10, the multi-target inhibitor DDP04-DS006 and DDP04-DS00601 of the present application both have obvious inhibition effect, which shows that different connection orders have obvious effect.

[1309] Table 11 Inhibition effect of DDP0401-DS001 series RNA inhibitors on C3, C5 mRNA in primary cynomolgus monkey hepatocytes

[1310] As can be seen from Table 11, compared with the delivery effect of the multi-target inhibitor and the inhibitor mixture, it can be seen that the multi-target inhibitor DDP0401 of the present application does not interfere with the inhibition effect of siRNA.

[1311] Table 12 Inhibition effect of DDP0402-DS007 series RNA inhibitors on C3, CFB mRNA in primary cynomolgus monkey hepatocytes

[1312] As can be seen from Table 12, compared with the delivery effect of the multi-target inhibitor and the inhibitor mixture, it can be seen that the multi-target inhibitor DDP04 of the present application does not interfere with the inhibition effect of siRNA, and even shows a better trend than the delivery effect of the inhibitor mixture.

[1313] Table 13 Inhibition effect of DDP0404-DS004, DDP0404-DSS004 series RNA inhibitors on APOC3, PCSK9 mRNA in primary cynomolgus monkey hepatocytes

[1314] As can be seen from Table 13, the DDP0404-DS004 and DDP0404-DSS004 sequences and the connecting carrier are the same, and only the modification of the 3' end nucleotide of the sense strand is different. It can be seen that the multi-target inhibitor DDP0404-DS004 and DDP0404-DSS004 of the present application do not interfere with the inhibitory effect of siRNA, and the two series even show a better trend than the delivery effect of the inhibitor mixture.

[1315] Table 14 Inhibitory effect of DDP0404-DS005 and DDP0404-DSS005 series RNA inhibitors on LPA and PCSK9 mRNA in primary cynomolgus monkey hepatocytes

[1316] The DDP0404-DS005 and DDP0404-DSS005 sequences and the connecting carrier are the same, and only the modification of the 3' end nucleotide of the sense strand is different. As can be seen from Table 14, comparing the delivery effect of the multi-target inhibitor and the inhibitor mixture, it can be seen that the multi-target inhibitor DDP0404-DS005 and DDP0404-DSS005 of the present application do not interfere with the inhibitory effect of siRNA, and even show a better trend than the delivery effect of the inhibitor mixture.

[1317] Table 15 Inhibitory effect of DDP0404-DS006 series RNA inhibitors on ANGPTL3 and PCSK9 mRNA in primary cynomolgus monkey hepatocytes

[1318] As can be seen from Table 15, comparing the delivery effect of the multi-target inhibitor and the inhibitor mixture, it can be seen that the multi-target inhibitor DDP0404 of the present application does not interfere with the inhibitory effect of siRNA, and even shows a better trend than the delivery effect of the inhibitor mixture.

[1319] Table 16 Inhibitory effect of DDP0404-DS006 series RNA inhibitors on ANGPTL3 and PCSK9 mRNA in primary cynomolgus monkey hepatocytes

[1320] The siRNA sequence modification and the structure of the connecting carrier in DDP0404-DS006 and DDP0404-DS00601 are the same, and only the connecting position of the orthogonal reaction group is different. As can be seen from Table 16, the multi-target inhibitor DDP0404-DS006 and DDP0404-DS00601 of the present application both have obvious inhibitory effect, indicating that different connecting positions have obvious effect.

[1321] Table 17 Inhibition effect of DDP0404-DS007 series RNA inhibitors on C3, CFB mRNA in primary cynomolgus monkey hepatocytes

[1322] As can be seen from Table 17, comparing the delivery effect of the multi-target inhibitor and the inhibitor mixture, it can be seen that the multi-target inhibitor DDP0404 of the present application does not interfere with the inhibition effect of siRNA, and even shows a better trend than the delivery effect of the inhibitor mixture.

[1323] Table 18 Inhibition effect of DDP0405-DS007 series RNA inhibitors on C3, CFB mRNA in primary cynomolgus monkey hepatocytes

[1324] As can be seen from Table 18, comparing the delivery effect of the multi-target inhibitor and the inhibitor mixture, it can be seen that the multi-target inhibitor DDP0405 of the present application does not interfere with the inhibition effect of siRNA, and even shows a better trend than the delivery effect of the inhibitor mixture.

[1325] Table 19 Inhibition effect of DDP0405 series single-target double-delivery RNA inhibitors on CFB mRNA in primary cynomolgus monkey hepatocytes

[1326] As can be seen from Table 19, comparing the delivery effect of the single-target double-delivery inhibitor and the single-delivery inhibitor at half the concentration, it can be seen that the single-target double-delivery inhibitor DDP0405 of the present application does not interfere with the inhibition effect of siRNA, and even shows a better trend than the delivery effect of the single-delivery inhibitor.

[1327] Table 20 Inhibition effect of DDP0405 series single-target double-delivery RNA inhibitors on C3 mRNA in primary cynomolgus monkey hepatocytes

[1328] As can be seen from Table 20, comparing the delivery effect of the single-target double-delivery inhibitor and the single-delivery inhibitor at half the concentration, it can be seen that the single-target double-delivery inhibitor DDP0405 of the present application does not interfere with the inhibition effect of siRNA, and even shows a better trend than the delivery effect of the single-delivery inhibitor.

[1329] Further verify the inhibition effect of the new chemical linker structure DDP05, DDP06, DDP07 based on the modification of DDP04 series. Based on the unchanged DPP05, DDP06, DDP07 chemical linker structure, the siRNA sequence is modified, the delivery carrier connection position is changed, and the DDP0501-DDP0504, DDP0601-DDP0604, DDP0601-DDP0604 series are obtained. The following data shows that the DPP05, DDP06, DDP07 series have obvious siRNA inhibition effect.

[1330] Table 21 Inhibition effect of DDP04, DDP05, DDP06, DDP07 series RNA inhibitors on C3, CFB mRNA in primary cynomolgus monkey hepatocytes

[1331] As can be seen from Table 21, by comparing the delivery effect of multi-target inhibitors and inhibitor mixtures, it can be seen that the multi-target inhibitors DDP04, DDP05, DDP06, DDP07 of the application do not interfere with the inhibition effect of siRNA, and even show a better trend than the delivery effect of the inhibitor mixture.

[1332] Table 22 Inhibition effect of DDP0402, DDP0502, DDP0602, DDP0702 series RNA inhibitors on C3, CFB mRNA in primary cynomolgus monkey hepatocytes

[1333] As can be seen from Table 22, by comparing the delivery effect of multi-target inhibitors and inhibitor mixtures, it can be seen that the multi-target inhibitors DDP0402, DDP0502, DDP0602, DDP0702 of the application do not interfere with the inhibition effect of siRNA, and even show a better trend than the delivery effect of the inhibitor mixture.

[1334] Further verify the inhibition effect of the new chemical linker structure DDP08, DDP09, DDP11 based on the modification of DDP04 series. The following data shows that the DDP08, DDP09, DDP11 series have obvious siRNA effect.

[1335] Table 23 Inhibition effect of DDP0404, DDP08 series RNA inhibitors on PCSK9, ANGPTL3 mRNA in primary cynomolgus monkey hepatocytes

[1336] As can be seen from Table 23, compared with the delivery effect of the multi-target inhibitor and the inhibitor mixture, the multi-target inhibitors (DDP0404, DDP08) of the present application do not interfere with the inhibitory effect of siRNA, and even show a better trend than the delivery effect of the inhibitor mixture.

[1337] Table 24 Inhibitory effect of DDP0404, DDP09 series RNA inhibitors on PCSK9, ANGPTL3 mRNA in primary cynomolgus monkey hepatocytes

[1338] As can be seen from Table 24, compared with the delivery effect of the multi-target inhibitor and the inhibitor mixture, the multi-target inhibitors (DDP0404, DDP09) of the present application do not interfere with the inhibitory effect of siRNA, and even show a better trend than the delivery effect of the inhibitor mixture.

[1339] Table 25 Inhibitory effect of DDP0404, DDP10 series RNA inhibitors on PCSK9, ANGPTL3 mRNA in primary cynomolgus monkey hepatocytes

[1340] As can be seen from Table 25, compared with the delivery effect of the multi-target inhibitor and the inhibitor mixture, the multi-target inhibitors (DDP0404, DDP10) of the present application do not interfere with the inhibitory effect of siRNA, and even show a better trend than the delivery effect of the inhibitor mixture.

[1341] Table 26 Inhibitory effect of DDP0404, DDP11 series RNA inhibitors on PCSK9, ANGPTL3 mRNA in primary cynomolgus monkey hepatocytes

[1342] As can be seen from Table 26, compared with the delivery effect of the multi-target inhibitor and the inhibitor mixture, the multi-target inhibitors (DDP0404, DDP11) of the present application do not interfere with the inhibitory effect of siRNA, and even show a better trend than the delivery effect of the inhibitor mixture.

[1343] 4.2 The RNA inhibitors of the present embodiment are selected from Table 1, and the inhibitory effect of HJY-0203 series RNA inhibitors is verified by BE(2)-C cell transfection, free uptake of cynomolgus monkey primary hepatocytes.

[1344] Experimental method of BE(2)-C cell transfection:

[1345] Cell plating: Take BE(2)-C cells in good growth condition and good fusion degree, discard the culture medium, add 5 mL PBS for rinsing once. After discarding the PBS, add 1 mL 0.25% trypsin solution, digest for 2 minutes at room temperature. Add 5 mL fresh culture medium to resuspend the cells, collect into a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, resuspend the cells with 2 mL fresh culture medium, count with Countstar Mira BF, and adjust the viable cells to a density of 2x10 5

[1346] Cell transfection: according to the test final concentration, dilute the siRNA sample with Opti-MEM to prepare a 20x intermediate solution. Prepare the RNAiMAX transfection reagent: configure the appropriate volume in a 15 ml centrifuge tube at a ratio of RNAiMAX:Opti-MEM = 1:50, vortex for 15 seconds, incubate at room temperature for 5 minutes. Take 25 μL of the diluted 20x compound intermediate solution of the corresponding concentration into a 96-well dilution plate, add 75 μL of diluted RNAiMAX / Optim-MEM to each well at the corresponding position, mix well and incubate for 15 minutes. Take 25 μL of the compound and RNAiMAX mixture from the dilution plate and add it to the corresponding position of the 96-well cell culture plate (the final volume is 125 μL per well), make 3 replicate wells for each concentration, mix gently, and place in a 5% CO2, 37°C incubator for 24 hours.

[1347] The experimental results are shown in Table 27:

[1348] Table 27 Inhibition effect of HJY-0203 series RNA inhibitors on APP and MAPT mRNA by BE(2)-C cell transfection

[1349] The 48h-TaqMan probe method was used to detect the inhibition effect of RNA inhibitors on APP and MAPT double target mRNA of primary cynomolgus monkey hepatocytes at concentrations of 3000 nM, 1000 nM, 300 nM and 100 nM. The test results are shown in Table 9.

[1350] Table 28 Inhibition effect of HJY-0203 series RNA inhibitors on APP and MAPT mRNA in primary cynomolgus monkey hepatocytes

[1351] ​From the results of Table 28, it can be seen that the structure of the HJY-0203 series of the application does not interfere with the inhibitory effect of siRNA, and even shows a better trend than the delivery effect of the inhibitor mixture.

[1352] The method for verifying the free uptake of cynomolgus monkey primary hepatocytes is as shown above, and the cynomolgus monkey primary hepatocytes freely take up siAPP and siMAPT, and the 72h-TaqMan probe method is used for detection. The cells are plated for 3h, and then the drugs are added. The inhibitory effect of the RNA inhibitor on the APP and MAPT double-target mRNA of the primary cynomolgus monkey hepatocytes is investigated at the concentrations of 1000nM, 250nM and 50nM. The test results obtained are shown in Table 10.

[1353] Table 29 Inhibitory effect of the HJY-0203 series of RNA inhibitors on APP and MAPT mRNA in primary cynomolgus monkey hepatocytes

[1354] From the results of Table 29, it can be seen that the inhibitory effect of the multi-target inhibitor HJY-0203 series of the application is generally better than that of the single-target or inhibitor mixture, and the performance of HJY-0203-DS026 and HJY-0203-DS027 is excellent, in particular, HJY-0203-DS027.

[1355] 4.3 Application of hyperlipidemic monkeys to evaluate the in vivo activity of RNA inhibitors

[1356] The RNA inhibitors of this example are selected from Table 1, and the inhibitory effect of the RNA inhibitors is verified by hyperlipidemic cynomolgus monkeys.

[1357] 4.3.1 Experimental process:

[1358] Male cynomolgus monkeys (TC≥6.2mmol / L, LDL-c≥4.1mmol / L, TG≥2mmol / L) over 8 years old, weighing more than 8 kg, are randomly divided into 6 groups, 2 in each group. D0 starts dosing, and the DDP siRNA inhibitor is administered at a dose of 18mg / kg, and the single-target siRNA inhibitor is administered at a dose of 9mg / kg. The administration method is subcutaneous injection, and the administration frequency is single administration. On Day 7, Day 14, Day 21, Day 28, Day 35, Day 42 and Day 49 after administration, blood samples are taken intravenously and the serum biomarkers and blood biochemical levels are detected. The biomarkers are target proteins such as PCSK9, APOC3, Lpa and ANGPTL3. The blood biochemistry includes TG (triglyceride), LDL-C (low-density lipoprotein), TC (total cholesterol).

[1359] 4.3.2 Experimental results

[1360] 4.3.2.1 PCSK9+APOC3 DDP0404-DS004 siRNA inhibitor has excellent in vivo effect

[1361] After administration of siRNA inhibitors, as shown in Figures 11-14: the expression levels of PCSK9 and APOC3 proteins in cynomolgus monkey serum were significantly decreased on the 7th day after administration, and the observation was observed up to the 49th day. DDP0404-DS004 siRNA inhibitor and single-target siRNA inhibitor (Kylo-08-DS00+Kylo-12-DS00) both have obvious inhibitory effect. DDP0404-DS004 siRNA and sing...

Claims

1. An RNA inhibitor chemically comprising a double-stranded small interfering nucleic acid directed against one or more targets, characterized in that: The specific structure is as follows: general formula Ia, Ib: X1and X2are orthogonal reactive groups, the orthogonal reactive groups include one or more of the following: amino, amide, carboxylic acid, azide, alkyne, propargyl, DBCO dibenzocyclooctyne, maleimide, aminooxy, N-hydroxysuccinimidyl NHS, PNP, TFP, PFP, bromo, aldehyde, carbonate, tosylate, tetrazine, trans-cyclooctene TCO, hydrazide, hydroxyl, disulfide and ortho-pyridyl disulfide groups, BCN bicyclo[6.1.0]nonyne, free or protected thiol; The Z a , Z b , Z c , Z d is a delivery ligand or H; k is an integer from 0 to 4; L1, L2, L3, L4 are a connecting chain or a bond, the connecting chain has the following general structure: -A-(B-A)n-: each B is independently a substituted or unsubstituted C1-10alkylene, C2-10alkenylene, C2-10alkynylene, C3-10cycloalkylene, C6-14arylene, heteroarylene, or heterocycloalkylene; Each A is an independent bond, -C(O)-, -C(O)O-, -C(O)NR 1b -,-NR 1b C(O)-,-(OCH2CH2) n -,-(CH2CH2O) n -,-(CH2OCH2) n -,-C(O)S-,-C(NR 1a )NR 1b -,-C(S)-,-C(S)O-,-C(S)NR 1b -,-C(R 1a )=NO-,-O-,-OC(O)O-,-OC(O)NR 1b -,-OC(O)S-,-OC(NR 1a )NR 1b -,-OC(S)O-,-OC(S)NR 1b -,-OS(O)-,-OS(O)2-,-OS(O)NR 1b -,-OS(O)2NR 1b -,-NR 1b -,-NR 1a C(O)NR 1b -,-NR 1a C(O)S-,-NR 1a C(NR 1d )NR 1b -,-NR 1a C(S)NR 1b -,-NR 1a S(O)NR 1b -,-NR 1a S(O)2NR 1b -,-P(O2)O-,-P(O)(S)O-,-S-,-SS-,-S(O)-,-S(O)2-,-S(O)NR 1b -, or -S(O)2NR 1b -; M can be selected from: substituted or unsubstituted alkyl groups, heteroalkyl groups, and can be selected from –C(O)–, –C(O)O–, –C(O)NR. 1b –,–NR 1b C(O)–,–(CH2)nC(O)NH(CH2)mNHC(O)(CH2)n–,–(OCH2CH2)n–,–(CH2CH2O)n–,–(CH2OCH2)n–,–(CH2)nC(O)NH(CH2CH2O)m(CH2)nNH C(O)(CH2)n–,–(CH2)nC(O)N(CH2)mCHC(O)NH(CH2)mNHC(O)CH(CH2)mNC(O)(CH2)n–,–C(O)S–,–C(NR 1a )NR 1b –,–C(S)–,–C(S)O–,–C(S)NR 1b –,–C(R 1a )=NO–,–O–,–OC(O)O–,–OC(O)NR 1b –,–OC(O)S–,–OC(NR 1a )NR 1b –,–OC(S)O–,–OC(S)NR 1b –,–OS(O)–,–OS(O)2–,–OS(O)NR 1b –,–OS(O)2NR 1b –,–NR 1b –,–NR1aC(O)NR 1b –,–NR 1a C(O)S–,–NR 1a C(NR 1d )NR 1b –,–NR 1a C(S)NR 1b –,–NR 1a S(O)NR 1b –,–NR 1a S(O)2NR 1b –,–P(O2)O–,–P(O)(S)O–,–S–,–S–S–,–S(O)–,–S(O)2–,–S(O)NR 1b –, or –S(O)2NR 1b –; each R 1a , and R 1b , and R 1c , and R 1d are independently: (i) hydrogen or deuterium; or (ii) substituted or unsubstituted C1-6alkyl, C1-6heteroalkyl, C2-6alkenyl, C2-6alkynyl, C3-10cycloalkyl, C6-14aryl, C7-15aralkyl, heteroaryl, or heterocyclyl; n is an integer from 0 to 10; m is an integer from 1 to 12; the delivery ligand includes one or more of the following: a ligand compound targeting ASGPR receptor, a ligand compound targeting αvβ6 integrin, a helper membrane permeable molecule, a polypeptide molecule, or an antibody molecule; the delivery ligand is conjugated to an end nucleotide of the siRNA, an internal nucleotide of the siRNA, or a connecting chain.

2. The RNA inhibitor of claim 1, which comprises a double-stranded small interfering nucleic acid targeting one or more targets in chemical structure, and characterized in that: I) concatenating the 5' end of the sense strand of the double-stranded small interfering nucleic acid with a delivery ligand conjugated to the 3' end or internal portion of the sense strand or the 3' end or internal portion of the antisense strand that is paired to the sense strand by the general formula (II); the general formula (IIa, lib) is shown below: II) concatenating the 3' ends of the sense strands of the double-stranded small interfering nucleic acids by general formula (IIIa, IIIb), which is conjugated to a delivery ligand at or in the 5' end of the sense strand or at or in the 5' end of the antisense strand which is complementary to the sense strand, as illustrated in the following schemes: III) conjugating the 3' end of the antisense strand of the double-stranded small interfering nucleic acid with a delivery ligand at the 5' end or internally of the sense strand that is paired to the antisense strand by a general formula (IVa, IVb); the general formula (IVa, IVb) is shown as follows: X1and X2are orthogonal reactive groups, the orthogonal reactive groups include one or more of the following: amino, amide, carboxylic acid, azide, alkyne, propargyl, DBCO dibenzocyclooctyne, maleimide, aminooxy, N-hydroxysuccinimidyl NHS, PNP, TFP, PFP, bromo, aldehyde, carbonate, tosylate, tetrazine, trans-cyclooctene TCO, hydrazide, hydroxyl, disulfide and ortho-pyridyl disulfide groups, BCN bicyclo[6.1.0]nonyne, free or protected thiol; ss is a sense strand, and as is an antisense strand; Z a , Z b , Z1, Z2, Z3, Z4, Z5, Z6are delivery ligands or H; L1, L2, L3, L4 are a connecting chain or a bond, the connecting chain has the following general structure: -A-(B-A)n-: each B is independently a substituted or unsubstituted C1-10alkylene, C2-10alkenylene, C2-10alkynylene, C3-10cycloalkylene, C6-14arylene, heteroarylene, or heterocycloalkylene; Each A is an independent bond, -C(O)-, -C(O)O-, -C(O)NR 1b -,-NR 1b C(O)-,-(OCH2CH2) n -,-(CH2CH2O) n -,-(CH2OCH2) n -,-C(O)S-,-C(NR 1a )NR 1b -,-C(S)-,-C(S)O-,-C(S)NR 1b -,-C(R 1a )=NO-,-O-,-OC(O)O-,-OC(O)NR 1b -,-OC(O)S-,-OC(NR 1a )NR 1b -,-OC(S)O-,-OC(S)NR 1b -,-OS(O)-,-OS(O)2-,-OS(O)NR 1b -,-OS(O)2NR 1b -,-NR 1b -,-NR 1a C(O)NR 1b -,-NR 1a C(O)S-,-NR 1a C(NR 1d )NR 1b -,-NR 1a C(S)NR 1b -,-NR 1a S(O)NR 1b -,-NR 1a S(O)2NR 1b -,-P(O2)O-,-P(O)(S)O-,-S-,-SS-,-S(O)-,-S(O)2-,-S(O)NR 1b -, or -S(O)2NR 1b -; M can be selected from: substituted or unsubstituted alkyl, heteroalkyl, can be selected from -C(O)-, -C(O)O-, -C(O)NR 1b , 1b C(O)-, -(CH2)nC(O)NH(CH2)mNHC(O)(CH2)n-, -(OCH2CH2)n-, -(CH2CH2O)n-, -(CH2OCH2)n-, -(CH2)nC(O)NH(CH2CH2O)m(CH2)nNHC(O)(CH2)n-, -(CH2)nC(O)N(CH2)mCHC(O)NH(CH2)mNHC(O)CH(CH2)mNC(O)(CH2)n-, -C(O)S-, -C(NR 1a )NR 1b , 1b C(S)-, -C(S)O-, -C(S)NR 1a , 1b C(R 1a ) = NO-, -O-, -OC(O)O-, -OC(O)NR 1b , 1b OC(S)O-, -OC(S)NR 1b , 1b OS(O)-, -OS(O)2-, -OS(O)NR 1b , 1b OS(O)2NR 1a , 1a NR 1d , 1b NR 1a , 1b NR 1a , 1b NR 1a , 1b P(O2)O-, -P(O)(S)O-, -S-, -S-S-, -S(O)-, -S(O)2-, -S(O)NR 1b , 1b S(O)2NR ; Each R 1a ,R 1b ,R 1c , and R 1d Independently: (i) hydrogen or deuterium; or substituted or unsubstituted (ii) C1-6 alkyl, C1-6 heteroalkyl, C2-6 alkenyl, C2-6 ynyl, C3-10 cycloalkyl, C6-14 aryl, C7-15 aryl, heteroaryl or heterocyclic. n is an integer from 0 to 10; m is an integer from 1 to 12; the delivery ligand includes one or more of the following: a ligand compound targeting ASGPR receptor, a ligand compound targeting αvβ6 integrin, a helper membrane permeable molecule, a polypeptide molecule, or an antibody molecule; the delivery ligand is conjugated to an end nucleotide of the siRNA, an internal nucleotide of the siRNA, or a connecting chain.

3. The RNA inhibitor of claim 1, wherein X1and X2are azide and DBCO, respectively, Compounds of the following specific formulae, Formulae Va, Vb, Via, VIb, Vic, VI d: wherein R1-R 10 are independently hydrogen, halogen, hydroxyl, or alkoxy, wherein the alkyl portion of alkoxy is C1, C2, C3, C4, C5, C7alkyl; Za, Zb, Zc, Zd are a delivery ligand or H; k is an integer from 0 to 4; L1, L2, L3, L4 are connecting chains or bonds, the connecting chains having the following general structure: -A-(B-A)n-; each B is independently substituted or unsubstituted C1-10alkylene, C2-10alkenylene, C2-10alkynylene, C3-10cycloalkylene, C6-14arylene, heteroarylene, or heterocycloalkylene; Each A is independently a bond, -C(O)-, -C(O)O-, -C(O)NR 1b -,-NR 1b C(O)-, -(OCH2CH2) n -,-(CH2CH2O) n -,-(CH2OCH2) n -,-C(O)S-,-C(NR 1a )NR 1b -,-C(S)-,-C(S)O-,-C(S)NR 1b -,-C(R 1a )=NO-,-O-,-OC(O)O-,-OC(O)NR 1b -,-OC(O)S-,-OC(NR 1a )NR 1b -,-OC(S)O-,-OC(S)NR 1b -,-OS(O)-,-OS(O)2-,-OS(O)NR 1b -,-OS(O)2NR 1b -,-NR 1b -,-NR 1a C(O)NR 1b -,-NR 1a C(O)S-,-NR 1a C(NR 1d )NR 1b -,-NR 1a C(S)NR 1b -,-NR 1a S(O)NR 1b -,-NR 1a S(O)2NR 1b -,-P(O2)O-,-P(O)(S)O-,-S-,-S-S-,-S(O)-,-S(O)2-,-S(O)NR 1b -, or -S(O)2NR 1b -; M can be selected from the group consisting of: substituted or unsubstituted alkyl, heteroalkyl, -C(O)-, -C(0)0-, -C(0)NR 1b , 1b C(O)-, -(CH2)nC(0)NH(CH2)mNHC(0)(CH2)n-, -(OCH2CH2)n-, -(CH2CH20)n-, -(CH20CH2)n-, -(CH2)nC(0)NH(CH2CH20)m(CH2)nNHC(0)(CH2)n-, -(CH2)nC(0)N(CH2)mCHC(0)NH(CH2)mNHC(0)CH(CH2)mNC(0)(CH2)n-, -C(0)S-, -C(S)-, -C(S)0-, -C(S)NR 1a , 1b 1b 1a 1b 1a 1b 1b 1b 1b 1b 1b 1a 1a 1d 1b 1a 1b 1a 1b 1a 1b , -P(02)0-, -P(0)(S)0-, -S-, -S-S-, -S(O)-, -S(0)2-, -S(0)NR 1b , 1b -;​​​​​​​​​​​​​​​​​​​​ wherein M is preferably selected from the group consisting of: Each R 1a ,R 1b ,R 1c , and R 1d Independently: (i) hydrogen or deuterium; or substituted or unsubstituted (ii) C1-6 alkyl, C1-6 heteroalkyl, C2-6 alkenyl, C2-6 ynyl, C3-10 cycloalkyl, C6-14 aryl, C7-15 aryl, heteroaryl or heterocyclic. n is an integer from 1 to 10; m is an integer from 1 to 12; the delivery ligand comprises one or more of a ligand compound targeting ASGPR receptor, a ligand compound targeting ανβ6 integrin, a helper transmembrane molecule, a polypeptide molecule, or an antibody molecule; the delivery ligand is conjugated to an end nucleotide of the siRNA, to an internal nucleotide of the siRNA, or to a connecting chain.

4. The RNA inhibitor of claim 1, wherein X1, X2 are each: thiol and olefin, and the specific compounds are as follows: compounds of general formula VII and VIII: wherein R1-R5 are independently hydrogen, halogen, hydroxyl, or alkoxy, wherein the alkyl portion of the alkoxy is C1, C2, C3, C4, C5, C7 alkyl; Za, Zb, Zc, Zd are delivery ligands or H; k is an integer from 0 to 4; L1, L2, L3, L4 are connecting chains or bonds, the connecting chains having the following general structure: -A-(B-A)n-; each B is independently substituted or unsubstituted C1-10alkylene, C2-10alkenylene, C2-10alkynylene, C3-10cycloalkylene, C6-14arylene, heteroarylene, or heterocycloalkylene; Each A is independently a bond, -C(O)-, -C(O)O-, -C(O)NR 1b -,-NR 1b C(O)-, -(OCH2CH2) n -,-(CH2CH2O) n -,-(CH2OCH2) n -,-C(O)S-,-C(NR 1a )NR 1b -,-C(S)-,-C(S)O-,-C(S)NR 1b -,-C(R 1a )=NO-,-O-,-OC(O)O-,-OC(O)NR 1b -,-OC(O)S-,-OC(NR 1a )NR 1b -,-OC(S)O-,-OC(S)NR 1b -,-OS(O)-,-OS(O)2-,-OS(O)NR 1b -,-OS(O)2NR 1b -,-NR 1b -,-NR 1a C(O)NR 1b -,-NR 1a C(O)S-,- NR 1a C(NR 1d )NR 1b -,-NR 1a C(S)NR 1b -,-NR 1a S(O)NR 1b -,-NR 1a S(O)2NR 1b -,-P(O2)O-,-P(O)(S)O-,-S-,-S-S-,-S(O)-,-S(O)2-,-S(O)NR 1b -,or-S(O)2NR 1b -; Each R 1a ,R 1b ,R 1c , and R 1d Independently: (i) hydrogen or deuterium; or substituted or unsubstituted (ii) C1-6 alkyl, C1-6 heteroalkyl, C2-6 alkenyl, C2-6 ynyl, C3-10 cycloalkyl, C6-14 aryl, C7-15 aryl, heteroaryl or heterocyclic. n is an integer from 1 to 10; the delivery ligand comprises one or more of a ligand compound targeting ASGPR receptor, a ligand compound targeting ανβ6 integrin, a helper transmembrane molecule, a polypeptide molecule, or an antibody molecule; the delivery ligand is conjugated to an end nucleotide of the siRNA, to an internal nucleotide of the siRNA, or to a connecting chain.

5. The RNA inhibitor of claim 1, wherein X1, X2 are BCN and azide, respectively, and are specifically the following compounds of general formula IX: wherein, Za, Zb, Zc, Zd are delivery ligands or H; k is an integer from 0 to 4; L1, L2, L3, L4 are connecting chains or bonds, the connecting chains having the following general structure: -A-(B-A)n-; each B is independently substituted or unsubstituted C1-10alkylene, C2-10alkenylene, C2-10alkynylene, C3-10cycloalkylene, C6-14arylene, heteroarylene, or heterocycloalkylene; Each A is independently a bond, -C(O)-, -C(O)O-, -C(O)NR 1b -,-NR 1b C(O)-, -(OCH2CH2) n -,-(CH2CH2O) n -,-(CH2OCH2) n -,-C(O)S-,-C(NR 1a )NR 1b -,-C(S)-,-C(S)O-,-C(S)NR 1b -,-C(R 1a ) = NO-,-O-,-OC(O)O-,-OC(O)NR 1b -,-OC(O)S-,-OC(NR 1a )NR 1b -,-OC(S)O-,-OC(S)NR 1b -,-OS(O)-,-OS(O)2-,-OS(O)NR 1b -,-OS(O)2NR 1b -,-NR 1b -,-NR 1a C(O)NR 1b -,-NR 1a C(O)S-,-NR 1a C(NR 1d )NR 1b -,-NR 1a C(S)NR 1b -,-NR 1a S(O)NR 1b -,-NR 1a S(O)2NR 1b -,-P(O2)O-,-P(O)(S)O-,-S-,-S-S-,-S(O)-,-S(O)2-,-S(O)NR 1b -, or -S(O)2NR 1b -; Each R 1a ,R 1b ,R 1c , and R 1d Independently: (i) hydrogen or deuterium; or substituted or unsubstituted (ii) C1-6 alkyl, C1-6 heteroalkyl, C2-6 alkenyl, C2-6 ynyl, C3-10 cycloalkyl, C6-14 aryl, C7-15 aryl, heteroaryl or heterocyclic. n is an integer from 1 to 10; the delivery ligand comprises one or more of a ligand compound targeting ASGPR receptor, a ligand compound targeting ανβ6 integrin, a helper transmembrane molecule, a polypeptide molecule, or an antibody molecule; the delivery ligand is conjugated to an end nucleotide of the siRNA, to an internal nucleotide of the siRNA, or to a connecting chain.

6. The RNA inhibitor of claim 1, wherein X1, X2 are each: azide and phosphine ester, and the specific compounds are as follows: general formula X: wherein R1-R3 are independently hydrogen, halogen, hydroxyl, or alkoxy, wherein the alkyl portion of the alkoxy is C1, C2, C3, C4, C5, C7 alkyl; Za, Zb, Zc, Zd are delivery ligands or H; k is an integer from 0 to 4; L1, L2, L3, L4 are connecting chains or bonds, the connecting chains having the following general structure: -A-(B-A)n-; each B is independently substituted or unsubstituted C1-10alkylene, C2-10 alkenylene, C2-10 alkynylene, C3-10 cycloalkylene, C6-14 arylene, heteroarylene, or heterocycloalkylene; Each A is independently a bond, -C(O)-, -C(O)O-, -C(O)NR 1b -,-NR 1b C(O)-, -(OCH2CH2) n -,-(CH2CH2O) n -,-(CH2OCH2) n -,-C(O)S-,-C(NR 1a )NR 1b -,-C(S)-,-C(S)O-,-C(S)NR 1b -,-C(R 1a ) = NO-,-O-,-OC(O)O-,-OC(O)NR 1b -,-OC(O)S-,-OC(NR 1a )NR 1b -,-OC(S)O-,-OC(S)NR 1b -,-OS(O)-,-OS(O)2-,-OS(O)NR 1b -,-OS(O)2NR 1b -,-NR 1b -,-NR 1a C(O)NR 1b -,-NR 1a C(O)S-,-NR 1a C(NR 1d )NR 1b -,-NR 1a C(S)NR 1b -,-NR 1a S(O)NR 1b -,-NR 1a S(O)2NR 1b -,-P(O2)O-,-P(O)(S)O-,-S-,-S-S-,-S(O)-,-S(O)2-,-S(O)NR 1b -, or -S(O)2NR 1b -; Each R 1a ,R 1b ,R 1c , and R 1d Independently: (i) hydrogen or deuterium; or substituted or unsubstituted (ii) C1-6 alkyl, C1-6 heteroalkyl, C2-6 alkenyl, C2-6 ynyl, C3-10 cycloalkyl, C6-14 aryl, C7-15 aryl, heteroaryl or heterocyclic. n is an integer from 1 to 10; the delivery ligand comprises one or more of a ligand compound targeting ASGPR receptor, a ligand compound targeting ανβ6 integrin, a helper transmembrane molecule, a polypeptide molecule, or an antibody molecule; the delivery ligand is conjugated to an end nucleotide of the siRNA, an internal nucleotide of the siRNA, or a linking chain.

7. The RNA inhibitor of claim 1, wherein The X1, X2 are respectively: TCO and tetrazine, the specific general formula is as follows: compound, general formula XI: wherein, Za, Zb, Zc, Zd are delivery ligands or H; k is an integer from 0 to 4; L1, L2, L3, L4 are linking chains or bonds, the linking chain having the structure of the general formula: -A-(B-A)n-: each B is independently substituted or unsubstituted C1-10alkylene, C2-10 alkenylene, C2-10 alkynylene, C3-10 cycloalkylene, C6-14 arylene, heteroarylene, or heterocycloalkylene; Each A is independently a bond, -C(O)-, -C(O)O-, -C(O)NR 1b -,-NR 1b C(O)-, -(OCH2CH2) n -,-(CH2CH2O) n -,-(CH2OCH2) n -,-C(O)S-,-C(NR 1a )NR 1b -,-C(S)-,-C(S)O-,-C(S)NR 1b -,-C(R 1a ) = NO-,-O-,-OC(O)O-,-OC(O)NR 1b -,-OC(O)S-,-OC(NR 1a )NR 1b -,-OC(S)O-,-OC(S)NR 1b -,-OS(O)-,-OS(O)2-,-OS(O)NR 1b -,-OS(O)2NR 1b -,-NR 1b -,-NR 1a C(O)NR 1b -,-NR 1a C(O)S-,-NR 1a C(NR 1d )NR 1b -,-NR 1a C(S)NR 1b -,-NR 1a S(O)NR 1b -,-NR 1a S(O)2NR 1b -,-P(O2)O-,-P(O)(S)O-,-S-,-S-S-,-S(O)-,-S(O)2-,-S(O)NR 1b -, or -S(O)2NR 1b -; Each R 1a ,R 1b ,R 1c , and R 1d Independently: (i) hydrogen or deuterium; or substituted or unsubstituted (ii) C1-6 alkyl, C1-6 heteroalkyl, C2-6 alkenyl, C2-6 ynyl, C3-10 cycloalkyl, C6-14 aryl, C7-15 aryl, heteroaryl or heterocyclic. n is an integer from 1 to 10; the delivery ligand comprises one or more of a ligand compound targeting ASGPR receptor, a ligand compound targeting ανβ6 integrin, a helper transmembrane molecule, a polypeptide molecule, or an antibody molecule; the delivery ligand is conjugated to an end nucleotide of the siRNA, an internal nucleotide of the siRNA, or a linking chain.

8. The RNA inhibitor for use according to any one of claims 1 to 7, wherein the RNA inhibitor is a double- stranded RNA molecule. Each L1, L2, L3, L4 is independently: wherein p is an integer from 0 to 200 and m is an integer from 0 to 12.

9. The RNA inhibitor for use according to claim 1 or 2, wherein the RNA inhibitor is a 5 modified oligonucleotide. The structure of the ligand compound targeting the ASGPR receptor is: Z R -L5-O-P(O)(O)-O-.

10. The RNA inhibitor of claim 1, wherein Z R The structure of the compound of formula (I) is shown below:

11. The RNA inhibitor of claim 4, wherein L5 is a linking chain, having the structure of the general formula: -A-(B-A)n-: each B is independently substituted or unsubstituted C1-10alkylene, C2-10 alkenylene, C2-10 alkynylene, C3-10 cycloalkylene, C6-14 arylene, heteroarylene, or heterocycloalkylene; each A is independently a bond, -C(O)-, -C(O)O-, -C(O)NR1b-, -NR1bC(O)-, -(OCH2CH2)n-, -(CH2CH2O)n-, -(CH2OCH2)n-, -C(O)S-, -C(NR1a)NR1b-, -C(S)-, -C(S)O-, -C(S)NR1b-, -C(R1a)=NO-, -O-, -OC(O)O-, -OC(O)NR1b-, -OC(O)S-, -OC(NR1a)NR1b-, -OC(S)O-, -OC(S)NR1b-, -OS(O)-, -OS(O)2-, -OS(O)NR1b-, -OS(O)2NR1b-, -NR1b-, -NR1aC(O)NR1b-, -NR1aC(O)S-, -NR1aC(NR1d)NR1b-, -NR1aC(S)NR1b-, -NR1aS(O)NR1b-, -NR1aS(O)2NR1b-, -P(O2)O-, -P(O)(S)O-, -S-, -S-S-, -S(O)-, -S(O)2-, -S(O)NR1b-, or -S(O)2NR1b-; each R1a, R1b, R1c, and R1d is independently: (i) hydrogen or deuterium; or (ii) substituted or unsubstituted C1-6alkyl, C1-6heteroalkyl, C2-6alkenyl, C2-6alkynyl, C3-10cycloalkyl, C6-14aryl, C7-15aralkyl, heteroaryl, or heterocyclyl; n is an integer from 1 to 10.

12. The RNA inhibitor of claim 6, wherein Each L5 independently is: wherein p is an integer from 0 to 200, and m is an integer from 0 to 12.

13. The RNA inhibiting agent of claim 4, wherein The structure of the ligand compound targeting ASGPR receptor is:

14. The RNA inhibitor for use according to claim 1 or 2, wherein the RNA inhibitor is a 5 modified oligonucleotide. The structure of the auxiliary membrane-spanning molecule is: wherein x is an integer from 1 to 10; each G is substituted or unsubstituted: C2-30alkyl, C2-30alkene, C2-30heteroalkyl, C2-30alkenyl, C2-30alkynyl, C2-30cycloalkyl, C2-30aryl, C2-30aryl, C2-30heteroaryl, or C2-30heterocyclyl; I is C or N; each L 4a、 L 4b having the structure -A-(B-A)n-: each B is independently a bond, substituted or unsubstituted C1-10alkylene, C2-10alkenylene, C2-10alkynylene, C3-10cycloalkylene, C6-14arylene, heteroarylene, or heterocyclylene; each A is independently a bond, -C(O)-, -C(O)O-, -C(O)NR1b-, -NR1bC(O)-, -(OCH2CH2)n-, -(CH2CH2O)n-, -(CH2OCH2)n-, -C(O)S-, -C(NR1a)NR1b-, -C(S)-, -C(S)O-, -C(S)NR1b-, -C(R1a)=NO-, -O-, -OC(O)O-, -OC(O)NR1b-, -OC(O)S-, -OC(NR1a)NR1b-, -OC(S)O-, -OC(S)NR1b-, -OS(O)-, -OS(O)2-, -OS(O)NR1b-, -OS(O)2NR1b-, -NR1b-, -NR1aC(O)NR1b-, -NR1aC(O)S-, -NR1aC(NR1d)NR1b-, -NR1aC(S)NR1b-, -NR1aS(O)NR1b-, -NR1aS(O)2NR1b-, -P(O2)O-, -P(O)(S)O-, -S-, -S-S-, -S(O)-, -S(O)2-, -S(O)NR1b-, or -S(O)2NR1b-; each R1a, R1b, R1c, and R1d is independently: (i) hydrogen or deuterium; or (ii) substituted or unsubstituted C1-6alkyl, C1-6heteroalkyl, C2-6alkenyl, C2-6alkynyl, C3-10cycloalkyl, C6-14aryl, C7-15aralkyl, heteroaryl, or heterocyclyl; n is an integer from 1 to 10.

15. The RNA inhibitor of claim 7, wherein The L 4a、 L 4b is: wherein p is an integer from 0 to 200 and m is an integer from 0 to 12.

16. The RNA inhibitor of claim 9, wherein The G structure comprises:

17. The RNA inhibitor of claim 1 or 2, wherein The oligonucleotide targets a target gene of C3, C5, CFB, SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene, APP, STAT3, Survivin gene, MAPT, PCSK9, ANGPTL3, APOC3, LPA, AGT, ALDH2, PNPLA3, KHK, HBV, LDHA, Factor VII, Eg5, TPX2, apoB, MYC, HPV, MKK4, CLAUDINE-1, SNCA, ACVRIC (ALK-7), Her2 / Neu gene, SID-1, MIG-12, MASP2, INFRSF12B, AT3, Inhibitor Factor VII, PTEN, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1 / 2 gene, PCNA (p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, Cyclin D gene, VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene, PKC gene, NFKB gene, Klf-1, bcl11a, serpinal, tmprss6, TTR, INHBE, CYP2A6, TM6SF2, Aha, IKK-b, CCR5, HAO1, SORT1 gene, XBP1 gene, Topoisomerase I gene, Topoisomerase II alpha gene, p73 gene, p21 (WAF1 / CIP1) gene, p27 (KIP1) gene, PPM1D gene, RAS gene, caveolin I gene, MIB I gene, MTAI gene, M68 gene, tumor suppressor gene, p53 tumor suppressor gene.

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